Human papillomavirus infections in women with clinical gynaecological diseases in southwest China

Abstract

The aim of this study was to determine the prevalence of human papillomavirus (HPV) infections in women with different gynaecological diseases (GDs) and ages in southwest China. With the assay of reverse dot blot, a total of 5544 cervical samples, including 1008 normal and 4536 abnormal specimens from women with GDs, were assayed for HPV detection. For the normal group, 20.2% (204/1008) tested positive for HPV, of which 40.2% (82/204) were high-risk (HR-HPV) genotypes. In the 4536 abnormal samples, 1569 women (34.6%) tested positive for HPV: 73.5% (1153/1569) had a single infection, 17.7% (277/1569) dual infection and 8.8% (138/1569) multiple HPV infection. Of 1569 HPV-positive specimens, 58.7% (921/1569) were infected with only HR-HPV genotypes. Significant differences in HPV infections were found among women of different ages (P < 0.01), number of pregnancies (P < 0.01), GDs (P < 0.01) and age at first sex (P < 0.01). In the present study, we found a high prevalence of HPV infection in women with GDs in southwest China. In addition to HPV types 16 and 18, a significant proportion of other HR-HPV genotypes were detected in this population.

Keywords

HPV human papillomavirus genotype reverse blot hybridization molecular epidemiology gynaecological disease China

INTRODUCTION

Human papillomaviruses (HPVs) are DNA viruses with a genome size of about 8000 base pairs.¹ There are more than 100 HPV types defined on the basis of DNA homology, of which more than 40 infect the anogenital tract.² Genital HPV types are typically divided into two groups according to their presumed oncogenic potential. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 are considered to be of high oncogenic risk (HR-HPV). The remaining genital types, such as types 6, 11, 42, 43 and 44, are considered of low or no oncogenic risk (LR-HPV).³ Infection by certain types of HPV is recognized as a causal and necessary factor for cervical cancer.^1,4 Cervical cancer is the second most common malignancy in women around the world and contributes to 9.8% of all female cancers.⁵ Other tumours related to HPV, such as anal, vaginal, vulvar, penile and oropharyngeal, represent an additional 0.7% of all cancer sites in both men and women, so that HPV is estimated to be responsible for 5.2% of all cancers worldwide.⁶ Subclinical and clinical genital warts, also known as condylomata acuminata, and virtually all squamous cell cancers of the anogenital tract, are caused by specific HPV types.^7,8

So far, various techniques have been used for HPV DNA detection: (i) direct probe methods, such as Southern blotting and in situ hybridization; (ii) signal amplification methods, such as the hybrid capture 2 (HC2) assay; and (iii) target amplification performed by a variety of polymerase chain reaction (PCR)-based techniques. For genotyping, PCRs are being followed by signal read-out methods, such as sequence analysis, restriction fragment length polymorphism (RFLP) analyses or hybridization with type-specific probes by different formats, such as membrane-based reverse line blot (RLB) assay and bead-based multiplex genotyping assay.⁹ Recently, several RLB assays based on different PCR protocols (MY09/11, SPF or GP5+/6+) have been developed and validated.^10–12 As the basic technique, membrane-based reverse dot blot has many advantages, such as high sensitivity and specificity. With one simple process, it can detect the HPV presence and also identify which type is present in the specimen.

A basic understanding of the HPV epidemiology is required in order to understand the role of various HPV types in the development of cervical cancer and to design effective vaccine strategies against the virus. Different populations may harbour varying HPV genotypes in the genital tract.¹³ The advent of molecular biological tools for HPV diagnosis has allowed us to identify HPV infection, differentiate HPV types and discriminate groups of the population with different risks of infection. HPV genotype prevalence varies in different populations.^14,15

Southwest China is an area with a higher cervical cancer mortality and a higher proportion of rural residents and women of lower socioeconomic status and has poorer health infrastructure when compared with eastern and southern China.^16,17 Thus far, there are few studies on the prevalence of HPV infections in women with gynaecological diseases (GDs) in southwest China investigating their prevalence of HPV genotypes. In the present study, samples collected from women with GDs in southwest China were assayed for HPV detection using the method of membrane-based RLB. The aim of this study was to estimate the prevalence of HPV infection in women with different GDs. It provides baseline data that will be accessible to insure that this population can be appropriately included in HPV vaccine trials in the future.

MATERIALS AND METHODS

Sample collection

Specimens were collected from women attending three hospitals (Sunshine Maternity Hospital, Chengdu Kangqiao Hospital and the Hospital Pertaining to the Procreation and Health Center of Sichuan Province) in southwest China from January 2005 to December 2007. Cervical samples were collected with a cervix brush from women attending for cervical cytology/Papanicolaou smears or sexually transmitted infection (STI) examinations. A history was taken and physical examination was performed on the patient. Cervical samples were divided into two groups: normal and abnormal cervical specimens. Eligible women with abnormal specimens (4536) included women aged >15 years who were residents of southwest China. Women were ineligible to participate in the study if they had a prior hysterectomy, normal cervical specimens, were pregnant at the examination or had blood-clotting disorders. At each visit, trained clinicians obtained exfoliated cervical cell samples for HPV DNA detection. All cervical brushes were taken and placed in a tube containing specimen transport medium (1 mL) and stored at −20°C until required for testing. Randomization was performed independently with the use of computer-generated random numbers. The women and the clinical personnel were unaware of the women's test assignments, and the laboratory technicians who performed the HPV tests had no personal information about the women. Lists with coded numbers specifying which samples should be tested were released to the virus laboratory only after the samples had arrived. At enrolment, a short survey was conducted, querying the patients about their social and demographic information as well as information on age, occupation, education, age at first sex, number of pregnancies and marriage status. As control group, 1008 cervical samples from women with normal cervices were also collected. Written informed consent was obtained from all participants of the study and ethical approval was received.

DNA detection from cervical samples

Upon receipt, cervical samples were processed for DNA extraction according to the protocol previously described by Gravitt et al. ¹⁰ The integrity of the extracted DNA was confirmed utilizing standard 1% agarose gel electrophoresis followed by staining with ethidium bromide. The DNA extracts were stored at −20°C until amplification by PCR.

PCR assay

HPV DNA was amplified by using L1 consensus primer. DNA quantity and integrity were monitored through amplification of part of the beta-globin gene in replicate tubes. Appropriate negative and positive controls were used to monitor the performance of the method.

The amplification system included 10× buffer, Mg²⁺, dNTP, ddH₂O, primers, Taq polymerase and template. And the amplification assay was: 50°C for 15 minutes; 95°C for 10 minutes; 40 cycles of 94°C for 30 seconds (denaturation); 52°C for 90 seconds (annealing); 72°C for 30 seconds (extension) and finally 72°C for 5 minutes.

Reverse blot hybridization

Type-specific biotinylated probes were designed according to the L1 gene and were anchored on a nylon membrane. Amplicons hybridized to probes were detected using streptavidin horseradish peroxidase-mediated colour precipitation. If a blue spot appeared in certain position on the membrane, it suggested that the specimen contained the corresponding HPV type. This method permits specific detection of 23 HPV genotypes: HPV 6, 11,16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, MM4 and 83.

Statistical analysis

Data analysis was performed on the SPSS statistical software (SPSS 13.0. SPSS Inc, Chicago, IL, USA). The univariate analysis of demographic and clinical differences were analysed using the chi-square test for frequencies and trend.

RESULTS

High prevalence of HPV infection in women with GDs in southwest China

The participants included in this study were sexually active with no previous histological diagnosis or treatment and were seeking cervical cancer screening. In this study, we screened 1008 normal cervical samples and 4536 abnormal ones, collected from women residing in southwest China, to detect HPV infection. The age of the normal group ranged from 17 to 65 years old, with an average age 30.1 years. Of these, 20.2% (204/1008) tested positive for HPV DNA, of which 40.2% (82/204) were infected with HR-HPV genotypes. The age of the abnormal group ranged from 15 to 67 years old, with an average age of 32.4 years at the time of testing. Of the 4536 abnormal samples, 1569 women (34.6%) tested positive for HPV DNA. Of the 1569 HPV-positive women, 73.5% (1153/1569) showed single HPV-type infection, 17.7% (277/1569) dual infection and 8.8% (138/1569) had multiple HPV-type infection, defined as a patient being infected with three or more HPV types at the same time (Figure 1).

Figure 1

The distribution of single, double, triple and multiple HPV infection among HPV-positive samples

Detection of uncommon HR-HPV types

Among all HPV-infected women with GDs in our study, 58.7% (921/1569) of the women were infected with HR-HPV types, 9.8% (153/1569) were infected with both HR- and LR-HPV types and 31.5% (495/1569) of the women tested positive for LR-HPV types only. The main HPV types identified in women with GDs in southwest China are shown in Table 1. In the population, HPV type 16 was present in the most cases (n = 654), followed by HPV 33 (n = 228), HPV 58 (n = 162) and HPV 18 (n = 99). Of the women infected with HR-HPV types, HPV 16/18 were collectively present in 661 women (42.1%). Overall, while the prevalence of HR-HPV genotypes is similar to the pattern identified in other populations, where HPV 16 was reported as the most common HR-HPV genotype, women in our study had a higher prevalence of certain other HR-HPV genotypes (notably HPV 33, 58 and 73).

Table 1

Incidence and frequency of high-risk (HR) and low-risk (LR) human papillomavirus (HPV) genotypes in women with gynaecological diseases

HPV genotype	Single infection	Multiple infection	Number of women (percentage of total women)	Percentage of total HPV infected women (n = 1569)
HR-HPV genotypes
16	374	280	654 (14.4%)	41.7
33	94	134	228 (5.0%)	14.5
58	69	93	162 (3.6%)	10.3
18	51	48	99 (2.2%)	6.3
59	41	27	68 (1.5%)	4.3
31	28	32	60 (1.3%)	3.8
73	29	31	60 (1.3%)	3.8
66	21	15	36 (0.8%)	2.3
52	18	15	33 (0.7%)	2.1
45	18	12	30 (0.7%)	1.9
51	5	13	18 (0.4%)	1.1
56	3	14	17 (0.4%)	1.1
LR-HPV genotypes
6	184	98	282 (6.2%)	18.0
11	146	112	258 (5.7%)	16.4
42	33	62	98 (2.2%)	6.2
43	21	25	46 (1.0%)	2.9
44	18	21	39 (0.9%)	2.5

Higher prevalence of HPV infection in younger women

In order to analyze the correlation between the frequency of HPV infection and various age groups, recruited women were divided into five groups according to their age. The prevalence of HPV infection was higher in younger women and showed a downward trend with increasing age from 35 years, and the HPV prevalence of the group aged between 25–34 years was higher than those below 25 years (χ ² = 42.350, P < 0.01) (Table 2).

Table 2

Prevalence of human papillomavirus (HPV) infection in specimens with selected characteristics

Characteristics	Positive	Negative	Total	HPV prevalence (%)	χ ²	P value
Ages (years)
15–24	144	233	377	38.2
25–34	765	1110	1875	40.8
35–44	399	772	1171	34.1	42.350	<0.001
45–54	203	481	684	29.7
>55	58	371	429	13.5
Total	1569	2967	4536	34.6
Gynaecological diseases
Cervicitis	456	908	1364	33.4
Cervical erosion	411	803	1214	33.9
Condylomata acuminata	360	257	617	58.3	47.655	<0.01
Others	342	999	1341	20.8
Total	1569	2967	4536	34.6
Normal group	204	804	1008	20.2
Ages at first sexual behaviour (years)
15–18	385	371	756	50.9
19–21	587	946	1533	38.3
22–24	428	1167	1595	26.8	49.183	<0.01
>25	169	483	652	25.9
Total	1569	2967	4536	34.6
Number of pregnancies
1	128	379	507	25.2
2	309	747	1056	29.1	19.116	<0.01
≥3	583	839	1422	41.0
Total	1020	1965	2985	34.2
Education
High school	498	885	1383	36.0
College	1071	2082	3153	34.1	0.590	>0.05
Total	1569	2967	4356	34.6

Different prevalence of HR- and LR-HPV genotypes in women with different GDs

Regarding the GDs, the patients with condylomata acuminata had the highest prevalence of HPV infection, and there was a significant difference among women with different GDs (χ ² = 47.655, P < 0.01) (Table 2). In addition, the distributions of HR- and LR-HPV genotypes in different GDs had significant patterns of HPV: in 456 HPV-positive cervicitis specimens, 309 (67.8%) carried only HR-HPV genotypes; in 411 HPV-positive cervical erosion specimens, 329 (80.0%) carried only HR-HPV genotypes, whereas of all 360 condylomata acuminata specimens, only 127 (35.3%) carried HR-HPV genotypes, as shown in Table 3.

Table 3

Distribution of high-risk (HR) and low-risk (LR) human papillomavirus (HPV) genotypes in the abnormal and normal cervical samples

	No. (%) of samples
Groups	Only HR-HPV genotypes	Both HR and LR-HPV genotypes	Only LR-HPV types	Total
Abnormal group
Cervicitis	309 (67.8)	32 (7.0)	115 (25.2)	456
Cervical erosion	329 (80.0)	61 (14.8)	21 (5.2)	411
Condylomata acuminate	127 (35.3)	17 (4.7)	216 (60.0)	360
Others diseases	156 (45.6)	43 (12.6)	143 (41.8)	342
Total	921 (58.7)	153 (9.8)	495 (31.5)	1569
Normal group	92 (45.1)	16 (7.8)	96 (47.1)	204

Higher prevalence of HPV infection in those who had first sex at an early age

HPV prevalence had significant associations with age at first sex and number of pregnancies. In the present study, with increasing age at first sex, the prevalence of HPV decreased (χ ² = 49.183, P < 0.01). Conversely, with increasing number of pregnancies, the prevalence of HPV increased (χ ² = 19.116, P < 0.01). In contrast, no association was observed between HPV and the education level (χ ² = 0.590, P > 0.05). The results are shown in Table 2.

The relationship between HPV infection and the time of sample collection

In the present study, we observed a correlation between HPV infection and the time of sample collection. For abnormal samples (n = 4536), the collection percentages ranged from 5.9% (267/4536) in April to 12.8% in July (582/4536). In our study, the prevalence of HPV-positive patients was higher in April and September, 42.3% (113/267) and 40.9% (168/411), respectively, followed by June (36.4%), July (36.1%) and May (36.1%), shown in Figure 1.

DISCUSSION

To our knowledge, this is the first study of the prevalence of cervical HPV among women with GDs in southwest China. In the present study, we collected 5544 cervical samples, of which 4536 samples were abnormal and 1008 normal samples were used as controls. In the abnormal group, all women had GDs, and 34.6% (1569/4536) women with GDs tested positive for HPV DNA. Of 1569 HPV-positive specimens, 921 (58.7%) were HR-HPV infections, which was higher than those in the normal group and also that reported from other regions of China.^18–20 The prevalence of HR-HPV for samples with cervicitis, cervical erosion and condylomata acuminata were 67.8%, 80.0%, and 35.3%, respectively. Epidemiological data suggest that 99.7% of cervical cancer tissue tests positive for HPV DNA; and HPV 16, 18, 31 and 45 are the most common types. About 50% cervical cancers are related to HPV 16.^21,22

The prevalence of HPV infection peaks in women aged in their early 20s, but because the infection is usually transient, the specificity of HPV screening is higher in women aged 35 or older than in younger women.^23–25 The age-specific incidence of cervical cancer peaks around the age of 40 years, which suggests that the efficacy of HPV testing should be maximal when it is performed on women between 30–40 years of age.²⁶ In order to analyze the correlation between the frequency of HPV infection and various age groups, recruited women were divided into five groups according to their age. We found that women aged 25–34 had the highest rate of HPV infection, and with increasing age, the rate decreased. The reason for this may be the higher frequency of sexual behaviour and lack of immunity. If infected with persistent HR-HPV, it may progress to malignant cervical lesions. Hence women, aged from 25–34 are perhaps the most important subjects for HPV testing. Our findings concur with other observations that the prevalence of HPV infection decreases with age. In women from Flanders with abnormal cytology, prevalences of 82% at age 22, 60% at age 47 and 52% at age 65 were found.²⁷ Also in our study, we found a predominance of HPV 16 infection. This is notable, since HPV 16 is considered high risk for cervical carcinoma.^28,29 Torres et al.'s³⁰ study found that young women were more likely to be infected with HR-HPV and develop cervical cancer with a poor prognosis.

Velema et al.'s³¹ study found that women who started sex before age 16 had a greater then two-fold increased incidence of cervical cancer. In our study, the prevalence of HPV among those who started sex aged between 15–18 was 50.9%, which was significantly higher than that of other age groups. With increasing numbers of pregnancies, the prevalence of HPV also increased accordingly. This may relate to impaired cervical immunity following induced abortions or confinements, increasing the risk of HPV infection.

In the present study, we observed a correlation between HPV infection and the time of year of sample collection. A Netherlands study (Hrushesky et al. ³²) also showed a seasonal correlation with HPV infection. According to their study, there was a positive correlation between summertime and HPV infectivity. Our sample collection percentages ranged from 5.9% in April to 12.8% for July. In our study, the prevalence of HPV-positive patients was higher in April and September (42.3% and 40.9%, respectively). We hypothesize that immunity of women in southwest China might decrease in the presence of wet weather and GDs, because it is the rainy season in April and September in southwest China. However, this hypothesis remains to be proven.

To effectively utilize HPV vaccines in women with GDs in southwest China, it is imperative to determine the HPV prevalence and genotypes of the population. Therefore, in addition to determining the frequency of HPV infection, we also investigated HPV genotypes in this population. In most studied populations, HPV 16 and HPV 18 were the most prevalent HR-HPV genotypes. In the present study, HPV 16 and HPV 18 were present in 48% of the HR-HPV infections. The remaining 52% of women were infected with other HR-HPV genotypes, such as HPV 33, our second most common genotype. HPV 33 has traditionally been found to be fully oncogenic in cell transformation and tumorigenesis assays.³³ One study³⁴ evaluated five major HPV types for their distributional changes along the spectrum of cervical carcinogenesis, which indicated their transformation activity clinically. In CIN1 and normal cervices, a parallel pattern of distributional proportion was noted, indicating a constant rate of forming CIN1 lesions in different HPV infections. In more severe cases of cervical neoplasia, changes in distributional proportion were characteristic in different HPV types. For example, in CIN2/3 versus normal and CIN2/3 versus CIN1 comparisons, the distributional proportion was stable in HPV 52, increased 2.1- to 5.4-fold in HPV 16, 58, and 33, and decreased 2- to 3.7-fold in HPV 18. Since the vaccines on the market include only HPV 16/18, ±6/11, the finding that HPV 33 is prevalent in our study may be important.

In conclusion, we found a high HPV prevalence and spectrum of HR-HPV genotypes in women with GDs; our study suggests that a substantial number of other HR-HPV types may contribute to cervical cancer in women of southwestern China.

Footnotes

ACKNOWLEDGEMENT

We thank Professor Zhu Ping for his critical comments on the manuscript and for his recommendations. The present work on the epidemiology of HPV infection in our laboratory is supported by Sunshine Maternity Hospital, Chengdu Kangqiao Hospital and the Hospital Pertaining to the Procreation and Health Center of Sichuan province.

References

Bosch

, Lorincz

, Muńoz

, The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002;55:244–65

Palefsky

, Holly

. Molecular virology and epidemiology of human papillomavirus and cervical cancer. Cancer Epidemiol Biomarkers Prev 1995;1:415–28

Munoz

, Bosch

, de Sanjose

, Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27

Hausen

. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002;2:342–50

Ferlay

, Bray

, Pisani

, GLOBOCAN 2002: cancer incidence, mortality and prevalence worldwide. IARC CancerBase, 2.0 edn. Lyon: IARC Press, 2004

Schiffman

, Castle

. Human papillomavirus: epidemiology and public health. Arch Pathol Lab Med 2003;127:930–34

Cutts

, Franceschi

, Goldie

, Human papillomavirus and HPV vaccines: a review. Bull World Health Organ 2007;85:719–26

Palefsky

. HPV infection in men. Dis Markers 2007;23:261–72

Hubbard

. Human papillomavirus testing methods. Arch Pathol Lab Med 2003;127:940–45

10.

Gravitt

, Peyton

, Apple

, Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method. J Clin Microbiol 1998;36:3020–27

11.

Kleter

, Doorn

, Schrauwen

, Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus. J Clin Microbiol 1999;37:2508–17

12.

van den Brule

, Pol

, Fransen-Daalmeijer

, GP5+/6+PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes. J Clin Microbiol 2002;40:779–87

13.

Sanjosé

, Diaz

, Castellsagué

, Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis 2007;7:453–59

14.

Wallin

, Wiklund

, Angström

, Type specific persistence of human papillomavirus DNA before the development of invasive cervical cancer. N Engl J Med 1999;341:1633–38

15.

Kjaer

, van den Brule

, Paull

, Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study. BMJ 2002;325:572–76

16.

Yang

, Huang

, Xiao

, Changes of mortality rate for cervical cancer during 1970's and 1990's periods in China. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2003;25:386–90

17.

Shi

, Wu

, Liu

, A cross-sectional study of distribution of human papillomavirus types in Shenzhen Women. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2006;28:832–36

18.

Zhao

, Li

, Ma

, Study of the association between human papillomavirus infection and cervical cancer in Xiangyuan county, Shanxi province. Chin J Epidemiol 2001;22:375–78

19.

Tan

, Zhao

, Liu

, The analysis of human papillomavirus infection of healthy women in Zhuhai. Maternal Child Health Care China 2005;20:1726–27

20.

Bao

, Li

, Smith

, Human papillomavirus type-distribution in the cervix of Chinese women: a meta-analysis. Int J STD AIDS 2008;19:106–11

21.

Burd

. Human papillomavirus and cervical cancer. Clin Micribiol Rev 2003;16:1–17

22.

Castle

. Beyond human papillomavirus the cervix, exogenous secondary factors, and the development of cervical precancer and cancer. Am Soc Colposcopy Cervical Pathol 2004;8:224–30

23.

Cuschieri

, Cubie

, Whitley

, Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population. J Clin Pathol 2004;57:68–72

24.

Evander

, Edlund

, Gustafsson

, Human papillomavirus infection is transient in young women: a population based cohort study. J Infect Dis 1995;171:1026–30

25.

Hagmar

, Kalantari

, Skyldberg

, Human papillomavirus in cell samples from Stockholm Gynecologic Health Screening. Acta Cytol 1995;39:741–45

26.

Cuzick

, Clavel

, Petry

, Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int J Cancer 2006;119:1095–101

27.

Sahebali

, Depuydt

, Segers

, Cervical cytological screening an human papillomavirus DNA testing in Flanderes. Acta Clin Belg 2003;58:211–19

28.

Berumen

, Ordonez

, Lazcano

, Asian-American variants of human papillomavirus 16 and risk for cervical cancer: a case-control study. J Natl Cancer Inst 2001;93:1325–30

29.

Franco

, Eliane

, Ferenczy

. Cervical cancer: epidemiology, prevention and the role of human papillomavirus infection. CMAJ 2001;164:1017–25

30.

Torres

, Rojo

, Torres

, Cervical cancer: current view of its epidemiology and risk factors. Gynecol Obstet Mex 2004;72:466–74

31.

Velema

, Ferrer

, Figueroa

, Burning wood in the kitchen increases the risk of cervical neoplasia in HPV-infected women in Honduras. Int J Cancer 2002;97:536–41

32.

Hrushesky

, Sothern

, Rietveld

, Sun exposure, sexual behavior and uterine cervical human papillomavirus. Int J Biometeorol 2006;50:167–73

33.

Pecoraro

, Morgan

, Defendi

. Differential effects of human papillomavirus type 6, 16, and 18 DNAs on immortalization and transformation of human cervical epithelial cells. Proc Natl Acad Sci USA 1989;86:563–67

34.

Ding

D-C

, Hsu

H-C

, Huang

R-L

, Type-specific distribution of HPV along the full spectrum of cervical carcinogenesis in Taiwan: An indication of viral oncogenic potential. Eur J Obstet Gynecol Reprod Biol 2008;140:245–51