Journal of Medical Virology 87:1248–1253 (2015)
Prevalence and Genotype Distribution of Human Papillomavirus Infection in Asymptomatic Women in Liaoning Province, China Hui Xue,1 Xuyong Lin,2 Tianren Li,1 Xiaoxia Yan,2 Kejun Guo,1 and Yi Zhang1* 1
Department of Gynecology, First Affiliated Hospital of China Medical University, Shenyang, China Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, China
2
Infection by human papillomavirus (HPV) is a necessary cause of cervical cancer. The purpose of this study was to investigate the prevalence and genotype distribution of HPV infection in Chinese women who were asymptomatic for cervical diseases. Cervical cytology samples were collected from 6479 asymptomatic Chinese women of Liaoning province, and tested for various HPV genotypes using a chip hybridization assay. HPV was found in 10.3% of all the asymptomatic women studied, with the prevalence of high risk HPV (HR HPV) and low risk HPV (LR HPV) being 9.5% and 1.1%, respectively. HPV genotypes 16, 52, and 58 were found the most frequently genotypes in the HR HPV positive women, and were present in 26.2%, 19.4% and 13.8%, respectively. A graph of HR HPV positive infection rates as a function of age is U-shaped, with a peak in women less than 30 years old and a second peak among women older than 50 years. Nearly half of the women infected with either HR HPV or LR HPV presented a normal looking cervix upon visual examination. The current study demonstrates that the epidemiology of HPV infection in asymptomatic Chinese women in Liaoning province is different from that in women from other regions, even from patients with cervical lesions in the same region. These findings could be used to guide the generation and design of an HPV vaccine for this population. J. Med. Virol. 87:1248–1253, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
human papillomavirus; prevalence; genotype; asymptomatic
INTRODUCTION HPV is a spherical double stranded DNA virus that belongs to the genus Papilloma vacuolating virus A C 2015 WILEY PERIODICALS, INC.
of the family Papovaviridae, and possesses a specific preference for human mucosa and skin [Handisurya et al., 2009]. A series of studies has established a relationship between HPV infection and cervical diseases. Infection with HR HPV is considered to be essential for the development of cervical cancer [Clifford et al., 2003] and is associated with the development of cervical intraepithelial neoplasia [Simonella et al., 2013]. At present, HPV detection is extensively used for cervical cancer screening owing to its high sensitivity compared with cytology smears [Moy et al., 2010]. Many studies have been performed to investigate the prevalence of HPV infection to estimate cervical lesions and, ultimately, for preventing the development of cervical cancer [Coutle´e et al., 2011; Ursu et al., 2011; Suthipintawong et al., 2011]. Relatively few surveys, however, have been conducted to determine the prevalence of HPV in generally asymptomatic women who have not been hospitalized. This study describes the results of a study conducted in Liaoning province that determined the prevalence and genotype distribution of HPV in 6479 asymptomatic Chinese women, to guide HPV vaccine application. MATERIALS AND METHODS Study Population Between January 2011 and July 2013, cervical cytology samples were collected from 6479 Chinese
Conflict of interest: We declare no potential conflicts of interest relevant to this article. Correspondence to: Yi Zhang, MD, PhD, Department of Gynecology, First Affiliated Hospital of China Medical University, 155 North Nanjing Street. Shenyang 110001, China. E-mail: [email protected] Accepted 26 June 2014 DOI 10.1002/jmv.24029 Published online 16 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
Prevalence and Genotype Distribution
women who underwent routine physical examinations at the Medical Examination Center of the First Affiliated Hospital of China Medical University in Liaoning province. None of the enrolled women had any previous history of HPV infection or any subjective symptoms for cervical diseases, such as leukorrhagia and bleeding after sexual intercourse. The ages of the participants ranged from 20 to 83 years and the median age was 42 years.
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Flow-Through Hybridization
Cervical specimens were collected with a disposable cervical cell collector (Hybribio, Chaozhou, Guangdong). The sampling head of the brush was placed into a 5-ml PreservCyt Solution vial (Hybribio, Chaozhou, Guangdong) and kept at 4˚C for no more than one week before DNA extraction. The samples were collected while the patients were not menstruating, not having been engaged in sexual activity for at least the previous 24 hr, and not having used an intravaginal drug for at least the previous 3 days.
Flow-through hybridization was performed using a microarray membrane on which HPV genotypespecific oligonucleotide probes were immobilized. The procedure was finished through a hybridization chamber (Hybribio, Chaozhou, Guangdong). The PCR products (25 ml) were heated at 95˚C for 5 min, then immediately placed into ice water for at least 2 min. The 1 ml preheated hybridization solution was added into the hybridization core and incubated for at least 2 min. The denatured DNA sample and 500 ml of preheated hybridization solution were mixed, placed on the chip, and incubated for more than 10 min, after which the flow-through hybridization was started. The flow-through hybridization allowed target DNA to hybridize and form duplex with the specific DNA probes on and within a membrane fiber. The instruments directed the target molecules toward the specific probes within the membrane fiber by downward pressure. After enzyme-labeled and coloration, the color point appeared, which matched with HPV genotype.
HPV DNA Extraction
Coloration
HPV DNA was extracted using the cell lysis kit (Hybribio, Chaozhou, Guangdong), according to the manufacturer’s instructions. Briefly, 500 ml of the liquid-based cytology specimen was centrifuged at 17,500 g for 1 min and the supernatant was discarded. A volume of 400 ml of solution I (the reagent consists of Tris, EDTA-2Na, SDS and so on) was added to the sample and incubated at 100˚C for 15 min. Solution II (isopropanol, 400 ml) was then added to the tube and centrifuged at 17,500 g for 5 min. After discarding the supernatant, centrifugation at 17,500 g was continued for another minute, and 60 ml of solution III (sterile water) was added after being placed for 2 min at room temperature.
The chip was blocked with 0.5 ml blocking solution (Tris-HCl Casein) twice and incubated with 0.5 ml enzyme solution (Tris, Streptavidin-AP conjugate) for 3.5 min at 25 ˚C. A volume of 0.5 ml of the chromogenic substrate NBT/BCIP was then added followed by three washes with solution B (NaCl, SDS) and one wash with 2 ml distilled water. The distinct visible bluish violet dot was regarded as HPV genotype positive. The process from DNA extraction to coloration was performed twice to confirm the HPV genotype.
Sample Collection
PCR Amplification HPV detection and genotyping were performed by DNA amplification, flow-through hybridization and a gene chip using the HPV GenoArray test kit (Hybribio, Chaozhou, Guangdong). The HPV GenoArray test kit uses MY09/11 primers to amplify a 450 bp region of the L1 gene. This assay can detect 15 HR HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68), and 6 LR HPV genotypes (HPV 6, 11, 42, 43, and 44, as well as CP8304). The reaction mixture for each participant contained 1 ml of DNA template, 0.75 ml DNA Taq and 23.25 ml PCR Mix in a total volume of 25 ml. The thermocycler was programmed as follows: 9 min at 95˚C; 40 cycles of 20 sec at 95˚C, 30 sec at 55˚C, 30 sec at 72˚C; and 5 min at 72˚C. Negative and positive controls were performed at the end of each PCR assay.
Statistical Analysis Statistical calculations were performed on the data using SPSS version 16.0. A chi-square test was used to check for the difference between data collected for different cervical histology groups and P values < 0.05 were considered significant. RESULTS HPV Prevalence in the Asymptomatic Women A total of 10.3% of the 6479 asymptomatic participants tested positive for one or more of the HPV DNA genotypes, with 9.2% (598/6479) testing positive for only HR HPV, 0.8% (51/6479) for only LR HPV, and 0.3% (17/6479) for both HR HPV and LR HPV (Table I). The data also revealed an escalating trend, where the prevalence of infection with HPV in 2013, at 14.2%, was significantly higher (P < 0.05) than that in 2011, which was 7.0%. Infections with multiple types of HPV were observed in 16.1% (107/666) of the HPV positive participants. Among these participants, 13.4% (89/666) were infected with multiple J. Med. Virol. DOI 10.1002/jmv
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Xue et al. TABLE I. The Prevalence of HPV From 2011 to 2013 HR HPV (þ)
Year
Total
2011 2012 2013 Total
1761 2751 1967 6479
Overall HPV (%) 124 263 279 666
(7.0) (9.6) (14.2) (10.3)
Single (%) 97 206 206 509
(5.5) (7.5) (10.5) (7.9)
LR HPV (þ)
Multiple (%) 14 28 47 89
(0.8) (1.0) (2.4) (1.4)
Single (%) 9 24 18 51
(0.5) (0.9) (0.9) (0.8)
Multiple (%) 0 1 (0.04) 0 1 (0.02)
Mixed infection 4 5 8 17
(0.2) (0.2) (0.4) (0.3)
Mixed infection of HR and LR HPV.
types of HR HPV, 2.6% (17/666) with both HR HPV and LR HPV (as mentioned above), and only one participant, that is 0.2%, was found to be infected with multiple types of LR HPV during the three years of the study. Of the 89 multiple HR HPV infections, 86.5% (77/89) were infected with two HPV genotypes, and 13.5% (12/89) were infected with three or more HPV genotypes. Two women were infected with six different HPV subtypes, and none with more than six. HPV Genotype Distribution The distribution of HPV genotypes in the enrolled women is summarized in Table II. Fourteen HR HPV genotypes were found, the most common being HPV16, detected in 26.2% of the 666 patients (Fig. 1). This genotype, together with the next two most common HR HPV genotypes, HPV52 and HPV58, were present in 59.4% of all the HR HPV infected women. Among LR HPV infections, HPV81 (CP8304) was the most common genotype, which was significantly
Cases in different year
HR-HPV 16 52 58 53 33 18 31 68 66 39 59 56 45 35 51 LR-HPV 81 (CP8304) 11 6 44 42 43
2011
2012
2013
36 10 20 17 13 8 6 10 6 2 0 2 0 1 0
45 56 37 33 14 20 11 14 14 10 7 5 3 2 2
80 53 28 32 20 18 23 15 11 11 8 4 6 6 4
3 5 5 0 0 0
19 4 4 3 0 0
9 9 6 1 1 0
J. Med. Virol. DOI 10.1002/jmv
Prevalence of HPV Infections as a Function of Age The overall prevalence of HPV infection among the women studied as a function of age yielded a U-shaped curve (Fig. 2). The highest HPV prevalence was observed in young women below 30 years of age, which was significantly higher than that in middleaged groups (those between 30 and 49 years old) (P < 0.05), but was not very significantly different from the HPV prevalence in women older than 50 years (P > 0.05) (Table III). Those women between the ages of 40 and 49 years showed the lowest HPV prevalence. Clinical Significance of Type-Specific HPV Infection The cervices of the women infected with HPV were visually examined in the present study (Table IV). A normal cervical appearance was the most common gross finding for women infected with either HR HPV or LR HPV, observed in 40.8% and 44.1% of the
TABLE II. The Distribution of HPV Genotypes
HPV genotype
more prevalent than HPV 11, HPV 6, HPV 44, and HPV 42. No participant was found to be infected with HPV 43 during the 3-year study.
Total (%) 161 119 85 82 47 46 40 39 31 23 15 11 9 9 6
(26.2) (19.4) (13.8) (13.3) (7.6) (7.5) (6.5) (6.3) (5.0) (3.7) (2.4) (1.8) (1.5) (1.5) (1.0)
31 (45.6) 18 (26.5) 15 (22.1) 4 (5.9) 1 (1.5) 0
Fig. 1. Age-specific prevalence in HPV-positive women.
Prevalence and Genotype Distribution
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Fig. 2. The distribution of HPV genotypes.
women in these groups, respectively. The second most common finding was cervical erosion, which was found in 35.3% of HR HPV positive women and 33.8% of those infected with LR HPV. The presence of a Nabothian cyst was the third most common finding and appeared in 30.4% of HR HPV positive women and 32.4% of LR HPV positive women. Cervical neoplasm was the most infrequent gross finding in both groups. DISCUSSION Cervical cancer is the second leading cause of cancer deaths among women worldwide. Yet, because of poor access to screening and treatment services, the vast majority of deaths occur in women living in
low and middle income countries, such as China [World Health Organization, 2013]. Epidemiological studies have clearly indicated that infection with oncogenic types of HPV is a necessary cause of cervical cancer [Mu~ noz et al., 2003]. Based on a study of women from Beijing, HR HPV infection rate was 39.9% for a cervical intraepithelial neoplasia I group, whereas the rate for a cervical intraepithelial neoplasia IIþ group (including cervical intraepithelial neoplasia II and III, early-stage squamous cervical carcinoma) was 88.6% [Li et al., 2010]. However, until now, no large-scale epidemiological study of the prevalence of HPV genotypes in asymptomatic Chinese females had been reported. In the current study, conducted in Liaoning province, PCR amplification was used to test for the presence of HPV DNA in a total of 6479 women asymptomatic for any cervical disease. The results showed that 10.3% of the participants were found to have acquired one or more genotypes of HPV, which was a little higher than the prevalence in an asymptomatic population in India [Srivastava et al., 2012]. The prevalence of HPV in the current study is obviously much lower than the HPV prevalence of 47.3% indicated from a previous study of women in the same general region of China [Sun et al., 2010]. This large difference in the reported HPV prevalence may be explained by noting that the participants enrolled in the current study, in contrast to those in the previous one, are from a general population of women without any symptoms of cervical lesions. The current results better represent the prevalence of HPV in healthy women who were not hospitalized, and thus contribute the most appropriate values for guiding HPV epidemiological surveys and vaccine applications. Furthermore, the results of the current study indicate that, at least in this region
TABLE III. Age-Specific Prevalence of HPV Infection HR-HPV Age
Total
< 30 30–39 40–49 50 Total
415 2123 2813 1128 6479
HPV Positive (%) 62 215 259 130 666
(14.9) (10.1) (9.2) (11.5) (10.3)
LR-HPV
Case
Prevalence (%)
Case
Prevalence (%)
57 205 232 121 615
13.7 9.7 8.3 10.7 9.5
5 16 27 9 68
1.2 0.8 1.3 0.9 1.1
TABLE IV. Relationship Between Visual Appearance of Cervix and HPV Infection HR-HPV
LR-HPV
Appearance
Total (%)
Case
Prevalence (%)
Case
Prevalence (%)
Clinical normal cervix Cervical erosion Nabothian cyst cervical neoplasm
275 232 22 204
251 217 30 187
40.8 35.3 4.9 30.4
30 23 2 22
44.1 33.8 2.9 32.4
(41.3) (34.8) (4.8) (30.6)
J. Med. Virol. DOI 10.1002/jmv
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of China, the prevalence of HPV positive infection is increasing, being significantly higher in 2013 than in 2011 (P < 0.05). Combating this sexually transmitted viral infection in asymptomatic females is thus becoming increasingly important to prevent the transmission of cervical diseases. The proposal that infection by multiple types of HPV increases the risk of developing cervical cancer remains controversial. It was found that women infected with HPV 16 alone were at similar or higher risk for cervical cancer than those infected with multiple types, including HPV 16 [Herrero et al., 2000]. Another study revealed that subjects infected with multiple HPV types had a 31.8-fold higher risk of developing cervical cancer, while infection with a single HPV type yielded a 19.9-fold increased risk compared with non-infected subjects [Lee et al., 2003]. In contrast, it was reported that multiple HPV type infection was correlated inversely with cervical lesion severity in Hong Kong, southern China [Chan et al., 2012]. In the present study, infection with multiple types of HPV accounted for 16.1% (107/666) of the HPV positive cases, a percentage that is lower than that found in other regions of China including the 25.5% in Guangdong [Chen et al., 2012], 38.5% in Shandong [Yuan et al., 2011], and 31.3% in Shenzhen [Li et al., 2006]. These differences could be due to differences in the enrolled population or detection methods. Since 2006, two prophylactic vaccines against the high-risk strains of HPV have been developed and approved in more than 100 countries. The distribution of HPV types, however, varies greatly across geographical regions worldwide [Clifford et al., 2003; Orozco-Colı´n et al., 2010], and in China, HPV vaccines are still under clinical trials [Liu et al., 2013]. The information regarding HPV genotype distribution in asymptomatic populations is very important for designing vaccines for women of the general population who have not noticed any symptoms of cervical lesions and have not gone to the hospital. In the present study, the most prevalent genotype in asymptomatic women from Liaoning province of China was found to be HPV 16, followed by successively lower prevalence of HPV 52, HPV 58, HPV 53, HPV 33, and HPV 18. Based on a metaanalysis, HPV 58 and 52 were the most prevalent HPV types in Chinese women [Li et al., 2013]. In the current study, the three major HPV types (HPV 16, HPV 52, and HPV 58) were present in 59.4% of all the women infected with HPV, which was notably consistent with the previous data [Li et al., 2013]. HPV 18 is a very common genotype in Western countries, but it is found only in a total of 1.8% of patients with specific cervical lesions (including cervicitis, cervical intraepithelial neoplasia and cervical cancer) and controls in Liaoning province, China [Sun et al., 2010]. In the current survey, HPV 18 was found in 7.5% of asymptomatic women, and is thus the sixth most common genotype. This result indiJ. Med. Virol. DOI 10.1002/jmv
Xue et al.
cates that the HPV 18 genotype should also be taken into account in the development of an HPV vaccine. HPV 81 (CP8304) was the most common LR HPV genotype in the current study, and was significantly more common than the other LR genotypes. It has been reported that the most prevalent genotypes in Chinese patients displaying genital warts were HPV 6 (41.3%) and HPV 11 (37.6%) [Chang et al., 2013]. These results show that HPV 81 (CP8304), while a very common infection in women in general, does not usually cause visible lesions such as general warts. HPV is the most common sexually transmitted virus [Abreu et al., 2012], and the current study indeed shows that its infection rate is relatively high for the ages at which women are most sexually active and able to bear children. A meta-analysis study of 46,900 European women showed the peak age for HPV infection to be less than 25 to 30 years, with steady declines thereafter [De Vuyst et al., 2009]. In the current study, the plot of HPV prevalence as a function of age is characterized by a U-shaped curve with a relatively high HPV prevalence at younger and older ages. The highest prevalence of HPV infection was 14.9% in women aged below 30 years, which may due to the fact that such women are more sexually active and more likely to have multiple partners than older women [Velicer et al., 2009; Zhao et al., 2012]. Nevertheless, the second peak of HPV infection was in women greater than 50 years of age, which may potentially be explained by reactivation of latent HPV infections in older women [Gonza´lez et al., 2010]. In China, large numbers of older women are unaware of the relationship between HPV and cervical cancer [Fu et al., 2010], and remain untested for HPV infection. The relatively high frequency of HPV infection in older women, as determined by the current study, reinforces the importance of their being screened for cervical diseases. The present survey also established relationships between type specific HPV infections and the visual appearance of the cervix in gynecological examinations. Since HPV infection is known to be the necessary cause of cervical cancer, it may often be assumed that oncogenic HPV infection would develop observable lesions upon gynecological, physical or colposcopic examination. In the current study, however, nearly half of the HPV positive women were observed to present a visually normal cervix. Perhaps some types of HPV produce no detectable alterations of the squamous epithelium in a subgroup of subjects, or some lesions are located in the endocervix and out of the reach of the visual evaluation [Jeronimo et al., 2007]. Another possible explanation of the lack association between HPV infection and visual lesions could be that persistent HPV infection progresses to cervical cancer or cervical intraepithelial neoplasia after a long latent period, while most women who acquire HPV infection are transiently infected [Wang et al., 2012]. Since not all the HPV infections are associated with visual changes of the cervical
Prevalence and Genotype Distribution
epithelium, women who present a normal appearing cervix should also be tested for HPV as part of cervical cancer screening. CONCLUSION HPV DNA detection has been used throughout the world for the screening of cervical cancer. The distribution of HPV genotypes in the given population has been the basis for the proper utilization of prophylactic vaccines. The information generated from the current study indicates that the prevalence and distribution of HPV genotypes in asymptomatic women were to a certain degree different from those in patients with cervical diseases, even when these two groups of women were from the same geographical region. These results provide important guidance for the generation and design of HPV vaccines tailored for the given population, and may even allow future evaluation of the impact of HPV vaccination programs among asymptomatic women in China. REFERENCES Abreu ALP, Souza RP, Gimenes F, Consolaro MEL. 2012. A review of methods for detect human Papillomavirus infection. Virol J 9:262. Chan PK, Cheung TH, Li WH, Yu MY, Chan MY, Yim SF, Ho WC, Yeung AC, Ho KM, Ng HK. 2012. Attribution of human papillomavirus types to cervical intraepithelial neoplasia and invasive cancers in Southern China. Int J Cancer 131:692–705. Chang L, Ci P, Shi J, Zhai K, Feng X, Colombara D, Wang W, Qiao Y, Chen W, Wu Y. 2013. Distribution of genital wart human papillomavirus genotypes in China: A multi-center study. J Med Virol 85:1765–1774. Chen Q, Luo ZY, Lin M, Lin QL, Chen CY, Yang C, Xie LX, Li H, Zheng JK, Yang LY, Ju GZ. 2012. Prevalence and genotype distribution of human papillomavirus infections in women attending hospitals in Chaozhou of Guangdong province. Asian Pac J Cancer Prev 13:1519–1524. Clifford GM, Smith JS, Plummer M, Mu~ noz N, Franceschi S. 2003. Human papillomavirus types in invasive cervical cancer worldwide: A meta-analysis. Br J Cancer 88:63–73. Coutle´e F, Ratnam S, Ramanakumar AV, Insinga RR, Bentley J, Escott N, Ghatage P, Koushik A, Ferenczy A, Franco EL. 2011. Distribution of human papillomavirus genotypes in cervical intraepithelial neoplasia and invasive cervical cancer in Canada. J Med Virol 83:1034–1041. De Vuyst H, Clifford G, Li N, Franceschi S. 2009. HPV infection in Europe. Eur J Cancer 45:2632–2639. Fu FH, Wang JB, Jiang Y. 2010. Survey on cognition of cervical cancer among women in community Beijing. China Cancer 19:643–646. Gonza´lez P, Hildesheim A, Rodrı´guez AC, Schiffman M, Porras C, Wacholder S, Pi~ neres AG, Pinto LA, Burk RD, Herrero R. 2010. Behavioral/lifestyle and immunologic factors associated with HPV infection among women older than 45 years. Cancer Epidemiol Biomarkers Prev 19:3044–3054. Handisurya A, Schellenbacher C, Kirnbauer R. 2009. Diseases caused by human papillomaviruses (HPV). J Dtsch Dermatol Ges 7:453–466. Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, Morales J, Balmaceda I, Greenberg MD, Alfaro M, Burk RD, Wacholder S, Plummer M, Schiffman M. 2000. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 92:464–474. Jeronimo J, Massad LS. Schiffman M; National Institutes of Health/American Society for Colposcopy and Cervical Pathology (NIH/ASCCP) Research Group 2007. Visual appearance of the uterine cervix: Correlation with human papillomavirus detection and type. Am J Obstet Gynecol 197:47.e1–48.
1253 Lee SA, Kang D, Seo SS, Jeong JK, Yoo KY, Jeon YT, Kim JW, Park NH, Kang SB, Lee HP, Song YS. 2003. Multiple HPV infection in cervical cancer screened by HPVDNA Chip. Cancer Lett 198:187–192. Li C, Wu M, Wang J, Zhang S, Zhu L, Pan J, Zhang W. 2010. A population-based study on the risks of cervical lesion and human papillomavirus infection among women in Beijing, People’s Republic of China. Cancer Epidemiol Biomarkers Prev 19:2655– 2664. Li J, Huang R, Schmidt JE, Qiao YL. 2013. Epidemiological features of human papillomavirus (hpv) infection among women living in Mainland China. Asian Pac J Cancer Prev 14:4015–4023. Li LK, Dai M, Clifford GM, Yao WQ, Arslan A, Li N, Shi JF, Snijders PJ, Meijer CJ, Qiao YL, Franceschi S. 2006. Human papillomavirus infection in Shenyang City, People’s Republic of China: A population-based study. Br J Cancer 95:1593–1597. Liu XY, Feng AH, Cui YM, Tobe RG. 2013. Prevention of human papillomavirus (HPV) infection and cervical cancer in China: How does HPV vaccination bring about benefits to Chinese women? Biosci Trends 7:159–167. Moy LM, Zhao FH, Li LY, Ma JF, Zhang QM, Chen F, Song Y, Hu SY, Balasubramanian A, Pan QJ, Koutsky L, Zhang WH, Lim JM, Qiao YL, Sellors JW. 2010. Human papillomavirus testing and cervical cytology in primary screening for cervical cancer among women in rural China: Comparison of sensitivity, specificity, and frequency of referral. Int J Cancer 127:646–656. Mu~ noz N, Bosch FX, de Sanjose´ S, Herrero R, Castellsague´ X, Shah KV, Snijders PJ, Meijer CJ. International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. 2003. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518–527. Orozco-Colı´n A, Carrillo-Garcı´a A, Me´ndez-Tenorio A, Ponce-deLeo n S, Mohar A, Maldonado-Rodrı´guez R, Guerra-Arias R, Flores-Gil O, Sotelo-Regil R, Lizano M. 2010. Geographical variation in human papillomavirus prevalence in Mexican women with normal cytology. Int J Infect Dis 14:e1082–1087. Simonella LM, Lewis H, Smith M, Neal H, Bromhead C, Canfell K. 2013. Type-specific oncogenic human papillomavirus infection in high grade cervical disease in New Zealand. BMC Infect Dis 13:114. Srivastava S, Gupta S, Roy JK. 2012. High prevalence of oncogenic HPV-16 in cervical smears of asymptomatic women of eastern Uttar Pradesh, India: A population-based study. J Biosci 37:63– 672. Sun ZR, Ji YH, Zhou WQ, Zhang SL, Jiang WG, Ruan Q. 2010. Characteristics of HPV prevalence among women in Liaoning province, China Int J Gynaecol Obstet 109:105–109. Suthipintawong C, Siriaunkgul S, Tungsinmunkong K, Pientong C, Ekalaksananan T, Karalak A, Kleebkaow P, Vinyuvat S, Triratanachat S, Khunamornpong S, Chongsuwanich T. 2011. Human papilloma virus prevalence, genotype distribution, and pattern of infection in Thai women. Asian Pac J Cancer Prev 12:853–856. Ursu RG, Onofriescu M, Nemescu D, Iancu LS. 2011. HPV prevalence and type distribution in women with or without cervical lesions in the Northeast region of Romania. Virol J 8:558. Velicer C, Zhu X, Vuocolo S, Liaw KL, Saah A. 2009. Prevalence and incidence of HPV genital infection in women. Sex Transm Dis 36:696–703. Wang S, Wei H, Wang N, Zhang S, Zhang Y, Ruan Q, Jiang W, Xiao Q, Luan X, Qian X, Zhang L, Gao X, Sun X. 2012. The prevalence and role of human papillomavirus genotypes in primary cervical screening in the northeast of China. BMC Cancer 12:160. World Health Organization. 2013. Comprehensive cervical cancer prevention and control: A healthier future for girls and women. WHO guidance note 1–112. Yuan X, Yang Y, Gu D, Liu H, Yang H, Wang M. 2011. Prevalence of human papillomavirus infection among women with and without normal cervical histology in Shandong Province, China Arch Gynecol Obstet 283:1385–1389. Zhao FH, Tiggelaar SM, Hu SY, Xu LN, Hong Y, Niyazi M, Gao XH, Ju LR, Zhang LQ, Feng XX, Duan XZ, Song XL, Wang J, Yang Y, Li CQ, Liu JH, Liu JH, Lu YB, Li L, Zhou Q, Liu JF, Zhao N, Schmidt JE, Qiao YL. 2012. A multi-center survey of age of sexual debut and sexual behavior in Chinese women: Suggestions for optimal age of human papillomavirus vaccination in China. Cancer Epidemiol 36:384–390.
J. Med. Virol. DOI 10.1002/jmv
Prevalence and Genotype Distribution of Human Papillomavirus Infection in Asymptomatic Women in Liaoning Province, China Hui Xue,1 Xuyong Lin,2 Tianren Li,1 Xiaoxia Yan,2 Kejun Guo,1 and Yi Zhang1* 1
Department of Gynecology, First Affiliated Hospital of China Medical University, Shenyang, China Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, China
2
Infection by human papillomavirus (HPV) is a necessary cause of cervical cancer. The purpose of this study was to investigate the prevalence and genotype distribution of HPV infection in Chinese women who were asymptomatic for cervical diseases. Cervical cytology samples were collected from 6479 asymptomatic Chinese women of Liaoning province, and tested for various HPV genotypes using a chip hybridization assay. HPV was found in 10.3% of all the asymptomatic women studied, with the prevalence of high risk HPV (HR HPV) and low risk HPV (LR HPV) being 9.5% and 1.1%, respectively. HPV genotypes 16, 52, and 58 were found the most frequently genotypes in the HR HPV positive women, and were present in 26.2%, 19.4% and 13.8%, respectively. A graph of HR HPV positive infection rates as a function of age is U-shaped, with a peak in women less than 30 years old and a second peak among women older than 50 years. Nearly half of the women infected with either HR HPV or LR HPV presented a normal looking cervix upon visual examination. The current study demonstrates that the epidemiology of HPV infection in asymptomatic Chinese women in Liaoning province is different from that in women from other regions, even from patients with cervical lesions in the same region. These findings could be used to guide the generation and design of an HPV vaccine for this population. J. Med. Virol. 87:1248–1253, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
human papillomavirus; prevalence; genotype; asymptomatic
INTRODUCTION HPV is a spherical double stranded DNA virus that belongs to the genus Papilloma vacuolating virus A C 2015 WILEY PERIODICALS, INC.
of the family Papovaviridae, and possesses a specific preference for human mucosa and skin [Handisurya et al., 2009]. A series of studies has established a relationship between HPV infection and cervical diseases. Infection with HR HPV is considered to be essential for the development of cervical cancer [Clifford et al., 2003] and is associated with the development of cervical intraepithelial neoplasia [Simonella et al., 2013]. At present, HPV detection is extensively used for cervical cancer screening owing to its high sensitivity compared with cytology smears [Moy et al., 2010]. Many studies have been performed to investigate the prevalence of HPV infection to estimate cervical lesions and, ultimately, for preventing the development of cervical cancer [Coutle´e et al., 2011; Ursu et al., 2011; Suthipintawong et al., 2011]. Relatively few surveys, however, have been conducted to determine the prevalence of HPV in generally asymptomatic women who have not been hospitalized. This study describes the results of a study conducted in Liaoning province that determined the prevalence and genotype distribution of HPV in 6479 asymptomatic Chinese women, to guide HPV vaccine application. MATERIALS AND METHODS Study Population Between January 2011 and July 2013, cervical cytology samples were collected from 6479 Chinese
Conflict of interest: We declare no potential conflicts of interest relevant to this article. Correspondence to: Yi Zhang, MD, PhD, Department of Gynecology, First Affiliated Hospital of China Medical University, 155 North Nanjing Street. Shenyang 110001, China. E-mail: [email protected] Accepted 26 June 2014 DOI 10.1002/jmv.24029 Published online 16 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
Prevalence and Genotype Distribution
women who underwent routine physical examinations at the Medical Examination Center of the First Affiliated Hospital of China Medical University in Liaoning province. None of the enrolled women had any previous history of HPV infection or any subjective symptoms for cervical diseases, such as leukorrhagia and bleeding after sexual intercourse. The ages of the participants ranged from 20 to 83 years and the median age was 42 years.
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Flow-Through Hybridization
Cervical specimens were collected with a disposable cervical cell collector (Hybribio, Chaozhou, Guangdong). The sampling head of the brush was placed into a 5-ml PreservCyt Solution vial (Hybribio, Chaozhou, Guangdong) and kept at 4˚C for no more than one week before DNA extraction. The samples were collected while the patients were not menstruating, not having been engaged in sexual activity for at least the previous 24 hr, and not having used an intravaginal drug for at least the previous 3 days.
Flow-through hybridization was performed using a microarray membrane on which HPV genotypespecific oligonucleotide probes were immobilized. The procedure was finished through a hybridization chamber (Hybribio, Chaozhou, Guangdong). The PCR products (25 ml) were heated at 95˚C for 5 min, then immediately placed into ice water for at least 2 min. The 1 ml preheated hybridization solution was added into the hybridization core and incubated for at least 2 min. The denatured DNA sample and 500 ml of preheated hybridization solution were mixed, placed on the chip, and incubated for more than 10 min, after which the flow-through hybridization was started. The flow-through hybridization allowed target DNA to hybridize and form duplex with the specific DNA probes on and within a membrane fiber. The instruments directed the target molecules toward the specific probes within the membrane fiber by downward pressure. After enzyme-labeled and coloration, the color point appeared, which matched with HPV genotype.
HPV DNA Extraction
Coloration
HPV DNA was extracted using the cell lysis kit (Hybribio, Chaozhou, Guangdong), according to the manufacturer’s instructions. Briefly, 500 ml of the liquid-based cytology specimen was centrifuged at 17,500 g for 1 min and the supernatant was discarded. A volume of 400 ml of solution I (the reagent consists of Tris, EDTA-2Na, SDS and so on) was added to the sample and incubated at 100˚C for 15 min. Solution II (isopropanol, 400 ml) was then added to the tube and centrifuged at 17,500 g for 5 min. After discarding the supernatant, centrifugation at 17,500 g was continued for another minute, and 60 ml of solution III (sterile water) was added after being placed for 2 min at room temperature.
The chip was blocked with 0.5 ml blocking solution (Tris-HCl Casein) twice and incubated with 0.5 ml enzyme solution (Tris, Streptavidin-AP conjugate) for 3.5 min at 25 ˚C. A volume of 0.5 ml of the chromogenic substrate NBT/BCIP was then added followed by three washes with solution B (NaCl, SDS) and one wash with 2 ml distilled water. The distinct visible bluish violet dot was regarded as HPV genotype positive. The process from DNA extraction to coloration was performed twice to confirm the HPV genotype.
Sample Collection
PCR Amplification HPV detection and genotyping were performed by DNA amplification, flow-through hybridization and a gene chip using the HPV GenoArray test kit (Hybribio, Chaozhou, Guangdong). The HPV GenoArray test kit uses MY09/11 primers to amplify a 450 bp region of the L1 gene. This assay can detect 15 HR HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68), and 6 LR HPV genotypes (HPV 6, 11, 42, 43, and 44, as well as CP8304). The reaction mixture for each participant contained 1 ml of DNA template, 0.75 ml DNA Taq and 23.25 ml PCR Mix in a total volume of 25 ml. The thermocycler was programmed as follows: 9 min at 95˚C; 40 cycles of 20 sec at 95˚C, 30 sec at 55˚C, 30 sec at 72˚C; and 5 min at 72˚C. Negative and positive controls were performed at the end of each PCR assay.
Statistical Analysis Statistical calculations were performed on the data using SPSS version 16.0. A chi-square test was used to check for the difference between data collected for different cervical histology groups and P values < 0.05 were considered significant. RESULTS HPV Prevalence in the Asymptomatic Women A total of 10.3% of the 6479 asymptomatic participants tested positive for one or more of the HPV DNA genotypes, with 9.2% (598/6479) testing positive for only HR HPV, 0.8% (51/6479) for only LR HPV, and 0.3% (17/6479) for both HR HPV and LR HPV (Table I). The data also revealed an escalating trend, where the prevalence of infection with HPV in 2013, at 14.2%, was significantly higher (P < 0.05) than that in 2011, which was 7.0%. Infections with multiple types of HPV were observed in 16.1% (107/666) of the HPV positive participants. Among these participants, 13.4% (89/666) were infected with multiple J. Med. Virol. DOI 10.1002/jmv
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Xue et al. TABLE I. The Prevalence of HPV From 2011 to 2013 HR HPV (þ)
Year
Total
2011 2012 2013 Total
1761 2751 1967 6479
Overall HPV (%) 124 263 279 666
(7.0) (9.6) (14.2) (10.3)
Single (%) 97 206 206 509
(5.5) (7.5) (10.5) (7.9)
LR HPV (þ)
Multiple (%) 14 28 47 89
(0.8) (1.0) (2.4) (1.4)
Single (%) 9 24 18 51
(0.5) (0.9) (0.9) (0.8)
Multiple (%) 0 1 (0.04) 0 1 (0.02)
Mixed infection 4 5 8 17
(0.2) (0.2) (0.4) (0.3)
Mixed infection of HR and LR HPV.
types of HR HPV, 2.6% (17/666) with both HR HPV and LR HPV (as mentioned above), and only one participant, that is 0.2%, was found to be infected with multiple types of LR HPV during the three years of the study. Of the 89 multiple HR HPV infections, 86.5% (77/89) were infected with two HPV genotypes, and 13.5% (12/89) were infected with three or more HPV genotypes. Two women were infected with six different HPV subtypes, and none with more than six. HPV Genotype Distribution The distribution of HPV genotypes in the enrolled women is summarized in Table II. Fourteen HR HPV genotypes were found, the most common being HPV16, detected in 26.2% of the 666 patients (Fig. 1). This genotype, together with the next two most common HR HPV genotypes, HPV52 and HPV58, were present in 59.4% of all the HR HPV infected women. Among LR HPV infections, HPV81 (CP8304) was the most common genotype, which was significantly
Cases in different year
HR-HPV 16 52 58 53 33 18 31 68 66 39 59 56 45 35 51 LR-HPV 81 (CP8304) 11 6 44 42 43
2011
2012
2013
36 10 20 17 13 8 6 10 6 2 0 2 0 1 0
45 56 37 33 14 20 11 14 14 10 7 5 3 2 2
80 53 28 32 20 18 23 15 11 11 8 4 6 6 4
3 5 5 0 0 0
19 4 4 3 0 0
9 9 6 1 1 0
J. Med. Virol. DOI 10.1002/jmv
Prevalence of HPV Infections as a Function of Age The overall prevalence of HPV infection among the women studied as a function of age yielded a U-shaped curve (Fig. 2). The highest HPV prevalence was observed in young women below 30 years of age, which was significantly higher than that in middleaged groups (those between 30 and 49 years old) (P < 0.05), but was not very significantly different from the HPV prevalence in women older than 50 years (P > 0.05) (Table III). Those women between the ages of 40 and 49 years showed the lowest HPV prevalence. Clinical Significance of Type-Specific HPV Infection The cervices of the women infected with HPV were visually examined in the present study (Table IV). A normal cervical appearance was the most common gross finding for women infected with either HR HPV or LR HPV, observed in 40.8% and 44.1% of the
TABLE II. The Distribution of HPV Genotypes
HPV genotype
more prevalent than HPV 11, HPV 6, HPV 44, and HPV 42. No participant was found to be infected with HPV 43 during the 3-year study.
Total (%) 161 119 85 82 47 46 40 39 31 23 15 11 9 9 6
(26.2) (19.4) (13.8) (13.3) (7.6) (7.5) (6.5) (6.3) (5.0) (3.7) (2.4) (1.8) (1.5) (1.5) (1.0)
31 (45.6) 18 (26.5) 15 (22.1) 4 (5.9) 1 (1.5) 0
Fig. 1. Age-specific prevalence in HPV-positive women.
Prevalence and Genotype Distribution
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Fig. 2. The distribution of HPV genotypes.
women in these groups, respectively. The second most common finding was cervical erosion, which was found in 35.3% of HR HPV positive women and 33.8% of those infected with LR HPV. The presence of a Nabothian cyst was the third most common finding and appeared in 30.4% of HR HPV positive women and 32.4% of LR HPV positive women. Cervical neoplasm was the most infrequent gross finding in both groups. DISCUSSION Cervical cancer is the second leading cause of cancer deaths among women worldwide. Yet, because of poor access to screening and treatment services, the vast majority of deaths occur in women living in
low and middle income countries, such as China [World Health Organization, 2013]. Epidemiological studies have clearly indicated that infection with oncogenic types of HPV is a necessary cause of cervical cancer [Mu~ noz et al., 2003]. Based on a study of women from Beijing, HR HPV infection rate was 39.9% for a cervical intraepithelial neoplasia I group, whereas the rate for a cervical intraepithelial neoplasia IIþ group (including cervical intraepithelial neoplasia II and III, early-stage squamous cervical carcinoma) was 88.6% [Li et al., 2010]. However, until now, no large-scale epidemiological study of the prevalence of HPV genotypes in asymptomatic Chinese females had been reported. In the current study, conducted in Liaoning province, PCR amplification was used to test for the presence of HPV DNA in a total of 6479 women asymptomatic for any cervical disease. The results showed that 10.3% of the participants were found to have acquired one or more genotypes of HPV, which was a little higher than the prevalence in an asymptomatic population in India [Srivastava et al., 2012]. The prevalence of HPV in the current study is obviously much lower than the HPV prevalence of 47.3% indicated from a previous study of women in the same general region of China [Sun et al., 2010]. This large difference in the reported HPV prevalence may be explained by noting that the participants enrolled in the current study, in contrast to those in the previous one, are from a general population of women without any symptoms of cervical lesions. The current results better represent the prevalence of HPV in healthy women who were not hospitalized, and thus contribute the most appropriate values for guiding HPV epidemiological surveys and vaccine applications. Furthermore, the results of the current study indicate that, at least in this region
TABLE III. Age-Specific Prevalence of HPV Infection HR-HPV Age
Total
< 30 30–39 40–49 50 Total
415 2123 2813 1128 6479
HPV Positive (%) 62 215 259 130 666
(14.9) (10.1) (9.2) (11.5) (10.3)
LR-HPV
Case
Prevalence (%)
Case
Prevalence (%)
57 205 232 121 615
13.7 9.7 8.3 10.7 9.5
5 16 27 9 68
1.2 0.8 1.3 0.9 1.1
TABLE IV. Relationship Between Visual Appearance of Cervix and HPV Infection HR-HPV
LR-HPV
Appearance
Total (%)
Case
Prevalence (%)
Case
Prevalence (%)
Clinical normal cervix Cervical erosion Nabothian cyst cervical neoplasm
275 232 22 204
251 217 30 187
40.8 35.3 4.9 30.4
30 23 2 22
44.1 33.8 2.9 32.4
(41.3) (34.8) (4.8) (30.6)
J. Med. Virol. DOI 10.1002/jmv
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of China, the prevalence of HPV positive infection is increasing, being significantly higher in 2013 than in 2011 (P < 0.05). Combating this sexually transmitted viral infection in asymptomatic females is thus becoming increasingly important to prevent the transmission of cervical diseases. The proposal that infection by multiple types of HPV increases the risk of developing cervical cancer remains controversial. It was found that women infected with HPV 16 alone were at similar or higher risk for cervical cancer than those infected with multiple types, including HPV 16 [Herrero et al., 2000]. Another study revealed that subjects infected with multiple HPV types had a 31.8-fold higher risk of developing cervical cancer, while infection with a single HPV type yielded a 19.9-fold increased risk compared with non-infected subjects [Lee et al., 2003]. In contrast, it was reported that multiple HPV type infection was correlated inversely with cervical lesion severity in Hong Kong, southern China [Chan et al., 2012]. In the present study, infection with multiple types of HPV accounted for 16.1% (107/666) of the HPV positive cases, a percentage that is lower than that found in other regions of China including the 25.5% in Guangdong [Chen et al., 2012], 38.5% in Shandong [Yuan et al., 2011], and 31.3% in Shenzhen [Li et al., 2006]. These differences could be due to differences in the enrolled population or detection methods. Since 2006, two prophylactic vaccines against the high-risk strains of HPV have been developed and approved in more than 100 countries. The distribution of HPV types, however, varies greatly across geographical regions worldwide [Clifford et al., 2003; Orozco-Colı´n et al., 2010], and in China, HPV vaccines are still under clinical trials [Liu et al., 2013]. The information regarding HPV genotype distribution in asymptomatic populations is very important for designing vaccines for women of the general population who have not noticed any symptoms of cervical lesions and have not gone to the hospital. In the present study, the most prevalent genotype in asymptomatic women from Liaoning province of China was found to be HPV 16, followed by successively lower prevalence of HPV 52, HPV 58, HPV 53, HPV 33, and HPV 18. Based on a metaanalysis, HPV 58 and 52 were the most prevalent HPV types in Chinese women [Li et al., 2013]. In the current study, the three major HPV types (HPV 16, HPV 52, and HPV 58) were present in 59.4% of all the women infected with HPV, which was notably consistent with the previous data [Li et al., 2013]. HPV 18 is a very common genotype in Western countries, but it is found only in a total of 1.8% of patients with specific cervical lesions (including cervicitis, cervical intraepithelial neoplasia and cervical cancer) and controls in Liaoning province, China [Sun et al., 2010]. In the current survey, HPV 18 was found in 7.5% of asymptomatic women, and is thus the sixth most common genotype. This result indiJ. Med. Virol. DOI 10.1002/jmv
Xue et al.
cates that the HPV 18 genotype should also be taken into account in the development of an HPV vaccine. HPV 81 (CP8304) was the most common LR HPV genotype in the current study, and was significantly more common than the other LR genotypes. It has been reported that the most prevalent genotypes in Chinese patients displaying genital warts were HPV 6 (41.3%) and HPV 11 (37.6%) [Chang et al., 2013]. These results show that HPV 81 (CP8304), while a very common infection in women in general, does not usually cause visible lesions such as general warts. HPV is the most common sexually transmitted virus [Abreu et al., 2012], and the current study indeed shows that its infection rate is relatively high for the ages at which women are most sexually active and able to bear children. A meta-analysis study of 46,900 European women showed the peak age for HPV infection to be less than 25 to 30 years, with steady declines thereafter [De Vuyst et al., 2009]. In the current study, the plot of HPV prevalence as a function of age is characterized by a U-shaped curve with a relatively high HPV prevalence at younger and older ages. The highest prevalence of HPV infection was 14.9% in women aged below 30 years, which may due to the fact that such women are more sexually active and more likely to have multiple partners than older women [Velicer et al., 2009; Zhao et al., 2012]. Nevertheless, the second peak of HPV infection was in women greater than 50 years of age, which may potentially be explained by reactivation of latent HPV infections in older women [Gonza´lez et al., 2010]. In China, large numbers of older women are unaware of the relationship between HPV and cervical cancer [Fu et al., 2010], and remain untested for HPV infection. The relatively high frequency of HPV infection in older women, as determined by the current study, reinforces the importance of their being screened for cervical diseases. The present survey also established relationships between type specific HPV infections and the visual appearance of the cervix in gynecological examinations. Since HPV infection is known to be the necessary cause of cervical cancer, it may often be assumed that oncogenic HPV infection would develop observable lesions upon gynecological, physical or colposcopic examination. In the current study, however, nearly half of the HPV positive women were observed to present a visually normal cervix. Perhaps some types of HPV produce no detectable alterations of the squamous epithelium in a subgroup of subjects, or some lesions are located in the endocervix and out of the reach of the visual evaluation [Jeronimo et al., 2007]. Another possible explanation of the lack association between HPV infection and visual lesions could be that persistent HPV infection progresses to cervical cancer or cervical intraepithelial neoplasia after a long latent period, while most women who acquire HPV infection are transiently infected [Wang et al., 2012]. Since not all the HPV infections are associated with visual changes of the cervical
Prevalence and Genotype Distribution
epithelium, women who present a normal appearing cervix should also be tested for HPV as part of cervical cancer screening. CONCLUSION HPV DNA detection has been used throughout the world for the screening of cervical cancer. The distribution of HPV genotypes in the given population has been the basis for the proper utilization of prophylactic vaccines. The information generated from the current study indicates that the prevalence and distribution of HPV genotypes in asymptomatic women were to a certain degree different from those in patients with cervical diseases, even when these two groups of women were from the same geographical region. These results provide important guidance for the generation and design of HPV vaccines tailored for the given population, and may even allow future evaluation of the impact of HPV vaccination programs among asymptomatic women in China. REFERENCES Abreu ALP, Souza RP, Gimenes F, Consolaro MEL. 2012. A review of methods for detect human Papillomavirus infection. Virol J 9:262. Chan PK, Cheung TH, Li WH, Yu MY, Chan MY, Yim SF, Ho WC, Yeung AC, Ho KM, Ng HK. 2012. Attribution of human papillomavirus types to cervical intraepithelial neoplasia and invasive cancers in Southern China. Int J Cancer 131:692–705. Chang L, Ci P, Shi J, Zhai K, Feng X, Colombara D, Wang W, Qiao Y, Chen W, Wu Y. 2013. Distribution of genital wart human papillomavirus genotypes in China: A multi-center study. J Med Virol 85:1765–1774. Chen Q, Luo ZY, Lin M, Lin QL, Chen CY, Yang C, Xie LX, Li H, Zheng JK, Yang LY, Ju GZ. 2012. Prevalence and genotype distribution of human papillomavirus infections in women attending hospitals in Chaozhou of Guangdong province. Asian Pac J Cancer Prev 13:1519–1524. Clifford GM, Smith JS, Plummer M, Mu~ noz N, Franceschi S. 2003. Human papillomavirus types in invasive cervical cancer worldwide: A meta-analysis. Br J Cancer 88:63–73. Coutle´e F, Ratnam S, Ramanakumar AV, Insinga RR, Bentley J, Escott N, Ghatage P, Koushik A, Ferenczy A, Franco EL. 2011. Distribution of human papillomavirus genotypes in cervical intraepithelial neoplasia and invasive cervical cancer in Canada. J Med Virol 83:1034–1041. De Vuyst H, Clifford G, Li N, Franceschi S. 2009. HPV infection in Europe. Eur J Cancer 45:2632–2639. Fu FH, Wang JB, Jiang Y. 2010. Survey on cognition of cervical cancer among women in community Beijing. China Cancer 19:643–646. Gonza´lez P, Hildesheim A, Rodrı´guez AC, Schiffman M, Porras C, Wacholder S, Pi~ neres AG, Pinto LA, Burk RD, Herrero R. 2010. Behavioral/lifestyle and immunologic factors associated with HPV infection among women older than 45 years. Cancer Epidemiol Biomarkers Prev 19:3044–3054. Handisurya A, Schellenbacher C, Kirnbauer R. 2009. Diseases caused by human papillomaviruses (HPV). J Dtsch Dermatol Ges 7:453–466. Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, Morales J, Balmaceda I, Greenberg MD, Alfaro M, Burk RD, Wacholder S, Plummer M, Schiffman M. 2000. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 92:464–474. Jeronimo J, Massad LS. Schiffman M; National Institutes of Health/American Society for Colposcopy and Cervical Pathology (NIH/ASCCP) Research Group 2007. Visual appearance of the uterine cervix: Correlation with human papillomavirus detection and type. Am J Obstet Gynecol 197:47.e1–48.
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J. Med. Virol. DOI 10.1002/jmv
