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Characteristics of HPV prevalence among women in Liaoning province, China
International Journal of Gynecology and Obstetrics 109 (2010) 105–109
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International Journal of Gynecology and Obstetrics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j g o
CLINICAL ARTICLE
Characteristics of HPV prevalence among women in Liaoning province, China Zheng-Rong Sun a, Yao-Hua Ji a, Wei-Qiang Zhou a, Shu-Lan Zhang b, Wei-Guo Jiang c, Qiang Ruan a,⁎ a b c
Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning, China Department of Gynecology and Obstetrics, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning, China Department of Pathology, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning, China
a r t i c l e
i n f o
Article history: Received 24 September 2009 Received in revised form 21 November 2009 Accepted 12 January 2010 Keywords: Cervical disease HPV genotype Prevalence
a b s t r a c t Objectives: To investigate the prevalence rates of specific human papillomavirus (HPV) types infecting women in Liaoning Province, China. Methods: Specimens from 4780 patients with cervical disease and 165 age-matched controls were tested for HPV genotypes using a chip hybridization assay. Results: The infection rates were 35.66% for patients with cervicitis, 54.61% for those with ASCUS, 64.14% for those with CIN, 83.76% for those with cervical cancer in situ, and 83.12% for those with invasive cervical cancer. The most common HPV genotype was HPV-16, followed by HPV-58, HPV-52, HPV-33, HPV-53, and HPV-31. There were 1529 single and 731 multiple infections among the 4780 patients. Single infections with high-risk genotypes were associated with various cervical diseases. HPV-16 was present in 399 of the patients with multiple infections. Conclusion: Compared with prevalence rates for other populations, the rates of specific HPV types infecting women are different in Liaoning Province of China. © 2010 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Epidemiological and experimental data have shown cervical cancer to be linked to infection with certain genotypes of human papillomavirus (HPV) [1]. Most HPV infections are transient, however, and the main risk factor for cervical intraepithelial neoplasia (CIN) is persistent cervical infection with a high-risk (HR) genotype [2]. These types include HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Others, which include types 6, 11, 40, 42, 43, 44, and 55, are associated with a relatively low risk (LR) of progression to CIN and invasive cervical cancer [3]. The main causes of invasive cervical cancer and its precursor lesions are HPV types 16 and 18 [4]. Treatments and vaccines have been developed to combat or prevent HPV [5]. Ideal vaccines will necessarily be local, however, targeting the HPV antigens the most prevalent in the regions in which they are distributed. A series of studies conducted worldwide, including in southern China, have assessed the prevalence of the various HPV types in different populations [6–8]. The present casecontrol study was conducted to determine which of the HPV types were the most prevalent among women with cervical disease in Liaoning province of northeastern China. 2. Materials and methods Between August 2007 and February 2009, 4780 women were tested for HPV DNA at the Affiliated Shengjing Hospital of China ⁎ Corresponding author. No 36, Sanhao Street, Heping District, Shenyang, China 110004. Tel.: +86 24 96615 13421; fax: +86 24 23892617. E-mail address: [email protected] (Q. Ruan).
Medical University, Shenyang, China. They were matched for age with 165 women with no obvious cervical disease who were seen routinely at the Medical Examination Center of the same hospital. Biopsy specimens were obtained from all women suspected of having cervical cancer, and cervical disease was confirmed by cytologic and/or histologic evaluation. The institutional ethics committee approved the study. As no personal information was to be published, we were exempted from requesting informed consent. Viral DNA was amplified and viral types detected using the HPV GenoArray test kit (HybriBio, Hong Kong) according to the manufacturer's instructions. This kit was chosen for its high rate (93.8%) of agreement with the commonly used Amplicor HPV test (Roche Molecular Systems, Alameda, California, USA) [9] and its good sensitivity, specificity, and reliability [10]. Its pool of HPV-specific primers and its microarray allow for DNA amplification and the detection of the following 21 HPV genotypes: 13 HR types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), 5 LR genotypes (6, 11, 42, 43, and 44), and 3 types common in China (53, 66, and CP8304). The cervical specimens were placed in the PreservCytR LBC medium (Cytyc, Bedford, MA, USA) and transported to the laboratory, where they were kept at temperatures between 2 °C and 8 °C until DNA extraction. The quality and quantity of the DNA extracted from the cervical cells were ascertained before amplification by polymerase chain reaction (PCR). The reaction mixture contained 1 μL of DNA template, 0.75 mM of magnesium chloride, 5 nmol of dNTPs, 12.5 pmoL of each primer, and 0.6 U of Taq polymerase in a total volume of 25 μL. The thermocycler was programmed as follows: 10 minutes at 20 °C, 9 minutes at 95 °C; 40 cycles of 20 seconds at 94 °C, 30 seconds at 55 °C, 30 seconds at 72 °C; and 5 minutes at 72 °C. Negative and positive controls were performed at the end of each PCR assay.
0020-7292/$ – see front matter © 2010 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2009.11.026
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3. Results
Table 1 Relationships between patient age and cervical lesions.a Age, y
Cervicitis
ASCUS
CIN
18–30 31–40 41–50 N 50
673 (67.91) 1139 (60.68) 900 (58.63) 207 (53.08)
64 83 105 33
233 564 405 106
(6.46) (4.42) (6.84) (8.46)
(23.51) (30.05) (26.38) (27.18)
CIS
CC
15 (1.51) 51 (2.72) 54 (3.51) 7 (1.80)
6 40 71 37
(0.61) (2.13) (4.63) (9.49)
Abbreviations: ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; CC, cervical cancer. a Values are given as number (percentage).
To detect HPV genotype-specific oligonucleotides, microarray hybridization was performed using a nylon membrane on which HPV genotype-specific oligonucleotide probes were immobilized. The PCR products were hybridized at 45 °C on such chips placed on the platform of a hybridization chamber (HybriBio). After blocking with confining fluid, the chips were washed 4 times in a 3× solution of SSPE (sodium chloride, sodium phosphate, and EDTA). The chromogenic substrate NBT/ BICP (nitroblue tetrozolium and 5-bromo-4-chloro-3-indoylphosphate) (Biosys Technology, Paris, France) was then added, followed by 3 washes in a 1× solution of SSPE and 1 wash with distilled water. Values were expressed as frequency and range and analyzed using STATA software (STATA, Cary, NC, USA). The Pearson χ2 test was used to check for correlations between presence of HPV and cell differentiation. P b 0.05 was considered significant. Continuous variables were categorized and a linear logistic regression model provided correlation coefficients as well as odds ratios (ORs) and 95% confidence intervals (CIs). In multivariate analysis, an unconditional linear logistic regression model provided maximum likelihood estimates of adjusted ORs and 95% CIs.
The analysis of demographic data revealed differences among the women with cervical cancer. Cancer prevalence increased with age. The highest prevalence, 9.49%, was found among women older than 50 years, compared with 4.63% for those aged 40 to 50 years, 2.13 % for those aged 30 to 40 years, and 0.61% for those aged 18 to 30 years (Table 1). The 4780 study patients and the 165 controls received cytologic and/or histologic evaluations, and histologic verification was carried out for those with abnormal cytologic results. Of the 4780 study patients, 2919 had cervicitis; 282 had atypical squamous cells of undetermined significance; 683 had grade 1 cervical intraepithelial neoplasia (CIN 1), 389 had CIN 2 lesions, and 236 had CIN 3 lesions; 117 had carcinoma in situ (CIS); and 154 had cervical cancer (CC). A total of 2260 women (47.28%) tested positive for HPV DNA. The most prevalent genotype (19.64%) was HPV-16, followed by HPV-58, HPV-52, HPV-33, HPV-53, and HPV-31. However, only 86 (1.80%) tested positive for HPV-18, which is a very common genotype in Western countries (Table 2). The 6 major HPV types were present in 79.16% of all infected women, and in 89.80% and 84.38%, respectively, of the women with CIS or CC. Moreover, 64.51% of the women with HPV DNA tested positive for HPV-16, HPV-58, and HPV-52 (Table 3). Infections with a single HPV type were detected in 26.05% of the study patients overall and 58.44% of the study patients with CC, compared with 12.73% of the controls. The rates of infection with a single HR type differed significantly between study patients and controls. The patients who had the lowest risk of being infected with a single HPV type were those with cervicitis (OR, 1.74; 95% CI, 1.09– 2.78). Infection with a single HR type was associated with various cervical conditions. The prevalence rates of single infection with
Table 2 Distribution of HPV genotypes among 165 women with no apparent lesions (controls) and 4780 patients with specific lesions.a HPV type
Controls
b
Cervicitis (n = 2919)
ASCUS (n = 282)
CIN (n = 1308)
CIS (n = 117)
CC (n = 154)
Total
High risk 16 58 52 33 31 68 18 39 35 59 51 56 45
18 (10.9) 2 (1.21) 3 (1.82) 1 (0.61) 1 (0.61) 0 1 (0.61) 0 0 1 (0.61) 0 0 0
334 (11.44) 201 (6.89) 155 (5.31) 99 (3.39) 63 (2.16) 45 (1.54) 41 (1.40) 40 (1.37) 36 (1.23) 22 (0.75) 18 (0.62) 16 (0.55) 4 (0.14)
57 (20.2) 33 (11.7) 20 (7.09) 12 (4.26) 8 (2.84) 5 (1.77) 9 (3.19) 5 (1.77) 5 (1.77) 7 (2.49) 3 (1.06) 2 (0.71) 0
392 (30.0) 147 (11.2) 117 (8.94) 97 (7.42) 59 (4.51) 33 (2.52) 25 (1.91) 27 (2.06) 7 (0.54) 15 (1.15) 15 (1.15) 8 (0.61) 7 (0.54)
71 (60.68) 18 (15.38) 3 (2.56) 7 (5.98) 10 (8.55) 2 (1.71) 4 (3.42) 0 1 (0.85) 0 0 0 0
85 (55.19) 12 (7.79) 11 (7.14) 5 (3.25) 5 (3.25) 4 (2.60) 7 (4.55) 4 (2.60) 0 1 (0.65) 1 (0.65) 3 (1.95) 3 (1.95)
939 (19.64) 401 (8.39) 306 (6.40) 220 (4.60) 145 (3.03) 89 (1.86) 86 (1.80) 76 (1.59) 49 (1.03) 45 (0.94) 37 (0.77) 29 (0.61) 14 (0.29)
4 (2.42) 2 (1.21) 1 (0.61)
73 (2.50) 59 (2.02) 39 (1.34)
13 (4.61) 6 (2.13) 7 (2.49)
50 (3.82) 48 (3.67) 28 (2.14)
9 (7.69) 5 (4.27) 6 (5.13)
7 (4.55) 10 (6.49) 6 (3.90)
152 (3.18) 128 (2.68) 86 (1.80)
1 (0.61) 2 (1.21) 0 1 (0.61) 1 (0.61) 36 (21.82)
69 (2.36) 50 (1.71) 18 (0.62) 5 (0.17) 4 (0.14) 1041 (35.66)
5 (1.77) 3 (1.06) 2 (0.71) 1 (0.35) 0 154 (54.61)
25 (1.91) 22 (1.68) 9 (0.69) 4 (0.31) 2 (0.15) 839 (64.14)
1 (0.85) 2 (1.71) 0 1 (0.85) 0 98 (83.76)
0 1 (0.65) 3 (1.95) 1 (0.65) 0 128 (83.12)
100 (2.09) 78 (1.63) 32 (0.67) 12 (0.25) 6 (0.13) 2260 (47.28)c
Intermediate risk 53 CP8304 66 Low risk 6 11 44 42 43 Total a b c
Abbreviations: Please see Table 1; values are given as number (percentage). These are the controls who tested positive for single or multiple infections. Of these 2260 patients, 1529 had a single infection and 731 had a multiple infection.
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Table 3 Major HPV types, alone or in combinations, present in the infected women (study patients and controls combined).a HPV types
Controls (n = 36)b
Cervicitis (n = 1041)
ASCUS (n = 154)
CIN (n = 839)
CIS (n = 98)
CC (n = 128)
Total (N = 2260)
16 16, 16, 16, 16,
18 21 22 25 26
334 622 690 749 791
57 84 90 98 104
392 571 634 663 698
71 (72.45) 79 (80.61) 84 (85.71) 87 (88.78) 88 (89.80)
85 (66.41) 102 (79.69) 102 (79.69) 106 (82.81) 108 (84.38)
939 1458 1600 1703 1789
a b
58, 52 58, 52, 33 58, 52, 33, 53 58, 52, 33, 53, 31
(50.00) (58.33) (61.11) (69.44) (72.22)
(32.08) (59.75) (66.28) (71.95) (75.98)
(37.01) (54.54) (58.44) (63.64) (67.53)
(46.72) (68.05) (75.57) (79.02) (83.19)
(41.55) (64.51) (70.80) (75.35) (79.16)
Abbreviations: Please see Table 1; values are given as number (percentage). These are the controls who tested positive for single or multiple infections.
LR types or types common in China were similar in all subgroups (Table 4). Infection with multiple HPV types was seen in 731 (32.35%) of the 2260 HPV-infected women, study patients and controls combined, or 15.29% of the 4780 study patients. Of these 731 patients, 399 were infected with HPV-16 as well as other HPV types. The rates of multiple infection were significantly higher in each subgroup of patients with a specific cervical disease than in the control group overall. The patients who had the lowest risk of having a multiple infection were those with cervicitis (OR, 3.33; 95% CI, 1.35–8.18). Infection with multiple HPV types may increase the risk of cervical disease (Table 4). Infection with an HR HPV genotype has been shown to be a major risk factor for cervical disease. Single infections with HPV-16 were significantly more prevalent among women with CC than among those with CIN (χ2 = 93.4) or among controls overall (χ2 = 50.8) (P b 0.05). A single infection with HPV-16 was the major risk factor for CC. Although a single infection with HPV-33 was relatively common among patients with CIN, it was rarely detected in those with CC. The prevalence of single infection with HPV-33 was null in the control group. In the study group, it was significantly higher among patients with CIN than among patients with CC (χ2 = 4.37, P b 0.05) (Table 5). 4. Discussion We conducted the present study using the HybriMax GenoArray System, a commercial oligonucleotide chip kit capable of detecting multiple types of HPV in cervical cells. The case-control design of the study allowed us to determine the distribution of HR HPV genotypes among women with cervical disease in northeastern China, as well as the prevalence rates of single and multiple infection with different
HPV types. The associations between HPV infection and grade of cervical lesion were also analyzed. A series of studies has established a relationship between HPV infection and cervical disease, and prevalence rates of HPV infection have been estimated for various populations in southern China [6–8]. We estimated an infection rate of 47.28% among women with cervicitis, preinvasive cervical disease, or invasive cervical cancer in Liaoning province in northeastern China. The most common genotype was HPV-16 in Liaoning province, followed by types 58, 52, 33, 53, and 31. One or more of these 6 genotypes were found in 79.16% of infected patients and accounted for 89.80% and 84.38% of infections, respectively, among women with CIS or CC. The 55.19% rate of HPV-16 infection we report for our CC subgroup is similar to the rate reported by Lo et al. [11]. In our study, HPV-58 was the second most common type, with an infection rate of 7.79% for our CC subgroup. In contrast, Chan et al. [12] reported that one-third of the women with CC in Hong Kong were positive for HPV-58, and similarly high rates have also been reported for Chinese populations living in Shanghai and Taiwan [7,8]. These studies suggest that HPV types 52 and 58 may play a more prominent role in the development of CC in Asia than HPV types 31, 33, and 45, which are more common on other continents [12]. Although HPV-18 has been found to be common in other regions of China and in other countries, we report a prevalence of 1.80% for our study patients and controls combined, and a prevalence of 4.55% for all women with CC, and these rates are lower than those reported for other parts of China. Lo et al. [11] reported a 14.8% rate of HPV-18 infection for Hong Kong women with CC. Another study also showed that HPV types 16 and 18 were the most common types in Indonesia, affecting, respectively, 41.9% and 37.8% of the women with CC [13]. The 35.66% HPV infection rate we are reporting for women with cervicitis is
Table 4 Distribution of single or multiple HPV infections among 165 women with no apparent lesions (controls) and 4780 patients with specific lesions.a HPV types
Controls (n = 165)
Cervicitis (n = 2919)
ASCUS (n = 282)
CIN (n = 1308)
CIS (n = 117)
CC (n = 154)
Total
64 8.28 4 1.13 2 0.557
(54.70) (4.61–14.86) (3.42) (0.30–4.31) (1.71) (0.11–2.92)
90 (58.44) 9.64 (5.51–16.86) 3 (1.95) 0.64 (0.15–2.71) 3 (1.95) 0.64 (0.15–2.71)
1245 (26.05)
28 (23.93) 10.08 (3.76–26.99)
32 (20.78) 8.40 (3.18–22.18)
Single infection High risk types
b
Common in China Low risk types
c
c
21 (12.73) 1.0 5 (3.03) 1.0 5 (3.03) 1.0
592 1.74 98 1.11 76 0.86
(20.28) (1.09–2.78) (3.36) (0.446–2.77) (2.60) (0.34–2.14)
71 (25.18) 2.31 (1.36–3.92) 13 (4.61) 1.55 (0.54–4.42) 5 (1.77) 0.58 (0.17–2.03)
428 3.34 55 1.41 25 0.62
(32.7) (2.08–5.35) (4.20) (0.55–3.56) (1.15) (0.24–1.65)
173 (3.62) 111 (2.32)
Multiple infection Low- and high-risk typesd a
5 (3.03) 1.0
275 (9.42) 3.33 (1.35–8.18)
65 (23.05) 9.59 (3.77–24.35)
331 (25.31) 10.84 (4.41–26.63)
731 (15.29)e
Abbreviations: Please see Table 1; values are given as number (percentage) and odds ratio (OR) and 95% confidence interval (CI). When the ORs for having a single HPV infection in the control group were compared with the corresponding ORs in each of these subgroups of patients, the differences were significantly different (χ2 = 5.63, P b 0.05 for the smallest difference). c When the ORs for having a single infection in the control group were compared with the corresponding ORs in each of these subgroups of patients, the differences were not significantly different (P N 0.05 in all cases). d When the ORs for having a multiple HPV infection in the control group were compared with the corresponding ORs in each of these subgroups of patients, the differences were significantly different (χ2 = 7.73, P b 0.05 for the smallest difference). e Among the 731 patients with multiple infections, 399 (54.58%) were infected by HPV-16 as well as other types. b
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Table 5 Distribution of 2 single HPV infections among controls and patients with CIN and CC.a HPV type
Controls (n = 165)
CIN (n = 1308)
CC (n = 154)
HPV-16 HPV-33
15 (9.09) 0
176 (13.46) 45 (3.44)c
68 (44.16)b 0
Abbreviations: CIN, cervical intraepithelial neoplasia; CC, cervical cancer. a Values are given as number (percentage). b The difference between the percentage of HPV-16 single infection in patients in the cervical cancer group and those in the CIN group or healthy women was statistically significant (χ2 = 93.4 and χ2 = 50.8, respectively, P b 0.05 for both). c The difference between the percentage of HPV-33 single infection in patients in the CIN group and those in the cervical cancer group or healthy women was statistically significant (χ2 = 4.37 for both, P b 0.05).
similar to the 33.1% rate (46 of 139 women) reported by Wu et al. [6] for Guangdong province in China. In our study, however, the rates of HPV infection for women with CIN 1, CIN 2, and CIN 3 (50.66%, 77.37% and 81.36%, respectively) are lower than those reported by these authors. Of the 154 study patients with CC, 128 (83.12%) were infected with HPV, and the difference with the controls was significant. This result is similar to the 89.9% (427 of 475 patients) reported for Guangdong province [6] and the 83% (88 of 106 patients) reported for southern China [7]. In this study, we analyzed infection with single HR HPV types in women with various cervical conditions, and the rates increased with lesion severity. This finding was consistent with previous findings [14]. Regarding infection with single LR HPV types or single HR types common in China, however, our findings did not match previous findings. The effect of multiple HPV infection on CC pathogenesis is unclear. Herrero et al. [15] found that women infected with HPV-16 alone were at similar or higher risk for CC than women infected with HPV-16 and another HPV type, but Lee et al. [16] reported an association between infection with multiple HPV types and increased risk of CC. Of the 4780 patients in the present study, 731 (15.29%) were infected with multiple HPV types; and of the 2260 women found to be infected with HPV in the study and control groups combined, 731 (32.35%) were infected with multiple HPV types. This is higher than in other studies, but the reported prevalence of multiple HPV infections varies widely, from 3.9% in Thailand [17], to 12.9% among women with squamous tumors in Peru [18], to 19.3% in Paraguay [19], to 32.2% in Korea [20], to 32% in Costa Rica [15], and to 34.3% in Mozambique [21]. These differences could be due to different rates of multiple infection in the various populations or to differences in the detection methods [22]. Of the 731 cases of multiple infections in our study, 399 involved HPV-16 plus other types. The difference between the rates of multiple HPV infection in the study patient and control groups was statistically significant. Infection with multiple types of HPV can increase the risk of cervical disease. It has been demonstrated that HPV infection is the main risk factor for the development of CC, and that HPV-16 is the genotype most frequently found in women with multiple HPV infections [23]. In our study, a single infection with HPV-16 was significantly more prevalent among women with CC than among women with CIN or among controls. Infection with HPV-16 alone can cause cervical cancer, and it is the main risk factor of CC in northern China. One of our findings is worth stressing. Whereas none of the 154 women with CC was infected with HPV-33, 45 of the 1308 women with CIN were. A single infection with HPV-33 therefore seemed more likely to cause CIN than CC in our population, and it will be worthwhile to test this observation in future studies. Information regarding HPV types found in given populations is very important for vaccine design. Based on our findings, and assuming that the efficacy of HPV vaccines containing HPV-16 is close to 100% [24], we could assume that about 55.19% of cases of CC would be prevented by using these vaccines in northeastern China. Our study provides basic prevalence data for HPV types in Liaoning province that can be
used to increase vaccine efficacy in that province. The prevalence of multiple infection with HPV-16, HPV-58, and HPV-52 was 79.69% for patients with CC. If the HPV vaccines also contained more HPV types, such as HPV-33, HPV-53, and HPV-31, the preventive efficacy should reach 84.38% for CC and 79.16% for any sort of precancerous lesion. An HPV vaccine for use in northeastern China should be designed against these 6 major HPV types first. Our findings further emphasize the importance of a vaccination program in northeastern China aimed at immunizing women before they become infected with HPV. Acknowledgment This work was supported by the National Natural Science Foundation of China (grant 30672248), the National Natural Science Foundation of China (grant 30770109), and the Project of Doctor Foundation of Liaoning Province in China (grant 20061039). Conflict of interest The authors declare that they have no conflict of interest. References [1] Van Tine BA, Kappes JC, Banerjee NS, Knops J, Lai L, Steenbergen RD, et al. Clonal selection for transcriptionally active viral oncogenes during progression to cancer. J Virol 2004;78(20):11172–86. [2] Schlecht NF, Kulaga S, Robitaille J, Ferreira S, Santos M, Miyamura RA, et al. Persistent human papillomavirus infection as a predictor of cervical intraepithelial neoplasia. JAMA 2001;286(24):3106–14. [3] Muñoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348(6):518–27. [4] zur Hausen H. Papillomavirus infections: a major cause of human cancers. Biochim Biophys Acta 1996;1288(2):55–78. [5] Alam S, Bromberg-White J, McLaughlin-Drubin M, Sen E, Bodily JM, Meyers C. Activity and therapeutic potential of ORI-1001 antisense oligonucleotide on human papillomavirus replication utilizing a model of dysplastic human epithelium. Anticancer Res 2005;25(2A):765–77. [6] Wu Y, Chen Y, Li L, Yu G, Zhang Y, He Y. Associations of high-risk HPV types and viral load with cervical cancer in China. J Clin Virol 2006;35(3):264–9. [7] Liu J, Rose B, Huang X, Liao G, Carter J, Wu X, et al. Comparative analysis of characteristics of women with cervical cancer in highversus low-incidence regions. Gynecol Oncol 2004;94(3):803–10. [8] Lai CH, Chao A, Chang CJ, Chao FY, Huang HJ, Hsueh S, et al. Host and viral factors in relation to clearance of human papillomavirus infection: a cohort study in Taiwan. Int J Cancer 2008;123(7):1685–92. [9] Grisaru D, Avidor B, Niv J, Marmor S, Almog B, Leibowitz C, et al. Pilot study of prevalence of high-risk human papillomavirus genotypes in Israeli Jewish women referred for colposcopic examination. J Clin Microbiol 2008;46(5):1602–4. [10] Carozzi F, Bisanzi S, Sani C, Zappa M, Cecchini S, Ciatto S, et al. Agreement between the Amplicor HPV test and the Hybrid Capture 2 assay in the detection of high-risk human papillomavirus and biopsy-confirmed high-grade cervical disease. J Clin Microbiol 2007;45(2):364–9. [11] Lo KW, Wong YF, Chan MK, Li JC, Poon JS, Wang VW, et al. Prevalence of human papillomavirus in cervical cancer: a multicenter study in China. Int J Cancer 2002;100(3):327–31. [12] Chan PK, Lam CW, Cheung TH, Li WW, Lo KW, Chan MY, et al. Association of human papillomavirus type 58 variant with the risk of cervical cancer. J Natl Cancer Inst 2002;94(16):1249–53. [13] Schellekens MC, Dijkman A, Aziz MF, Siregar B, Cornain S, Kolkman-Uljee S, et al. Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. Gynecol Oncol 2004;93(1):49–53. [14] Sun CA, Lai HC, Chang CC, Neih S, Yu CP, Chu TY. The significance of human papillomavirus viral load in prediction of histologic severity and size of squamous intraepithelial lesions of uterine cervix. Gynecol Oncol 2001;83(1):95–9. [15] Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, Morales J, et al. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 2000;92(6):464–74. [16] Lee SA, Kang D, Seo SS, Jeong JK, Yoo KY, Jeon YT, et al. Multiple HPV infection in cervical cancer screened by HPVDNAChip. Cancer Lett 2003;198(2):187–92. [17] Chichareon S, Herrero R, Muñoz N, Bosch FX, Jacobs MV, Deacon J, et al. Risk factors for cervical cancer in Thailand: a case-control study. J Natl Cancer Inst 1998;90(1): 50–7. [18] Santos C, Muñoz N, Klug S, Almonte M, Guerrero I, Alvarez M, et al. HPV types and cofactors causing cervical cancer in Peru. Br J Cancer 2001;85(7):966–71. [19] Rolón PA, Smith JS, Muñoz N, Klug SJ, Herrero R, Bosch X, et al. Human papillomavirus infection and invasive cervical cancer in Paraguay. Int J Cancer 2000;85(4):486–91. [20] Huang HJ, Huang SL, Lin CY, Lin RW, Chao FY, Chen MY, et al. Human papillomavirus genotyping by a polymerase chain reaction-based genechip method in cervical
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International Journal of Gynecology and Obstetrics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j g o
CLINICAL ARTICLE
Characteristics of HPV prevalence among women in Liaoning province, China Zheng-Rong Sun a, Yao-Hua Ji a, Wei-Qiang Zhou a, Shu-Lan Zhang b, Wei-Guo Jiang c, Qiang Ruan a,⁎ a b c
Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning, China Department of Gynecology and Obstetrics, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning, China Department of Pathology, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning, China
a r t i c l e
i n f o
Article history: Received 24 September 2009 Received in revised form 21 November 2009 Accepted 12 January 2010 Keywords: Cervical disease HPV genotype Prevalence
a b s t r a c t Objectives: To investigate the prevalence rates of specific human papillomavirus (HPV) types infecting women in Liaoning Province, China. Methods: Specimens from 4780 patients with cervical disease and 165 age-matched controls were tested for HPV genotypes using a chip hybridization assay. Results: The infection rates were 35.66% for patients with cervicitis, 54.61% for those with ASCUS, 64.14% for those with CIN, 83.76% for those with cervical cancer in situ, and 83.12% for those with invasive cervical cancer. The most common HPV genotype was HPV-16, followed by HPV-58, HPV-52, HPV-33, HPV-53, and HPV-31. There were 1529 single and 731 multiple infections among the 4780 patients. Single infections with high-risk genotypes were associated with various cervical diseases. HPV-16 was present in 399 of the patients with multiple infections. Conclusion: Compared with prevalence rates for other populations, the rates of specific HPV types infecting women are different in Liaoning Province of China. © 2010 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Epidemiological and experimental data have shown cervical cancer to be linked to infection with certain genotypes of human papillomavirus (HPV) [1]. Most HPV infections are transient, however, and the main risk factor for cervical intraepithelial neoplasia (CIN) is persistent cervical infection with a high-risk (HR) genotype [2]. These types include HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Others, which include types 6, 11, 40, 42, 43, 44, and 55, are associated with a relatively low risk (LR) of progression to CIN and invasive cervical cancer [3]. The main causes of invasive cervical cancer and its precursor lesions are HPV types 16 and 18 [4]. Treatments and vaccines have been developed to combat or prevent HPV [5]. Ideal vaccines will necessarily be local, however, targeting the HPV antigens the most prevalent in the regions in which they are distributed. A series of studies conducted worldwide, including in southern China, have assessed the prevalence of the various HPV types in different populations [6–8]. The present casecontrol study was conducted to determine which of the HPV types were the most prevalent among women with cervical disease in Liaoning province of northeastern China. 2. Materials and methods Between August 2007 and February 2009, 4780 women were tested for HPV DNA at the Affiliated Shengjing Hospital of China ⁎ Corresponding author. No 36, Sanhao Street, Heping District, Shenyang, China 110004. Tel.: +86 24 96615 13421; fax: +86 24 23892617. E-mail address: [email protected] (Q. Ruan).
Medical University, Shenyang, China. They were matched for age with 165 women with no obvious cervical disease who were seen routinely at the Medical Examination Center of the same hospital. Biopsy specimens were obtained from all women suspected of having cervical cancer, and cervical disease was confirmed by cytologic and/or histologic evaluation. The institutional ethics committee approved the study. As no personal information was to be published, we were exempted from requesting informed consent. Viral DNA was amplified and viral types detected using the HPV GenoArray test kit (HybriBio, Hong Kong) according to the manufacturer's instructions. This kit was chosen for its high rate (93.8%) of agreement with the commonly used Amplicor HPV test (Roche Molecular Systems, Alameda, California, USA) [9] and its good sensitivity, specificity, and reliability [10]. Its pool of HPV-specific primers and its microarray allow for DNA amplification and the detection of the following 21 HPV genotypes: 13 HR types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), 5 LR genotypes (6, 11, 42, 43, and 44), and 3 types common in China (53, 66, and CP8304). The cervical specimens were placed in the PreservCytR LBC medium (Cytyc, Bedford, MA, USA) and transported to the laboratory, where they were kept at temperatures between 2 °C and 8 °C until DNA extraction. The quality and quantity of the DNA extracted from the cervical cells were ascertained before amplification by polymerase chain reaction (PCR). The reaction mixture contained 1 μL of DNA template, 0.75 mM of magnesium chloride, 5 nmol of dNTPs, 12.5 pmoL of each primer, and 0.6 U of Taq polymerase in a total volume of 25 μL. The thermocycler was programmed as follows: 10 minutes at 20 °C, 9 minutes at 95 °C; 40 cycles of 20 seconds at 94 °C, 30 seconds at 55 °C, 30 seconds at 72 °C; and 5 minutes at 72 °C. Negative and positive controls were performed at the end of each PCR assay.
0020-7292/$ – see front matter © 2010 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2009.11.026
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3. Results
Table 1 Relationships between patient age and cervical lesions.a Age, y
Cervicitis
ASCUS
CIN
18–30 31–40 41–50 N 50
673 (67.91) 1139 (60.68) 900 (58.63) 207 (53.08)
64 83 105 33
233 564 405 106
(6.46) (4.42) (6.84) (8.46)
(23.51) (30.05) (26.38) (27.18)
CIS
CC
15 (1.51) 51 (2.72) 54 (3.51) 7 (1.80)
6 40 71 37
(0.61) (2.13) (4.63) (9.49)
Abbreviations: ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; CC, cervical cancer. a Values are given as number (percentage).
To detect HPV genotype-specific oligonucleotides, microarray hybridization was performed using a nylon membrane on which HPV genotype-specific oligonucleotide probes were immobilized. The PCR products were hybridized at 45 °C on such chips placed on the platform of a hybridization chamber (HybriBio). After blocking with confining fluid, the chips were washed 4 times in a 3× solution of SSPE (sodium chloride, sodium phosphate, and EDTA). The chromogenic substrate NBT/ BICP (nitroblue tetrozolium and 5-bromo-4-chloro-3-indoylphosphate) (Biosys Technology, Paris, France) was then added, followed by 3 washes in a 1× solution of SSPE and 1 wash with distilled water. Values were expressed as frequency and range and analyzed using STATA software (STATA, Cary, NC, USA). The Pearson χ2 test was used to check for correlations between presence of HPV and cell differentiation. P b 0.05 was considered significant. Continuous variables were categorized and a linear logistic regression model provided correlation coefficients as well as odds ratios (ORs) and 95% confidence intervals (CIs). In multivariate analysis, an unconditional linear logistic regression model provided maximum likelihood estimates of adjusted ORs and 95% CIs.
The analysis of demographic data revealed differences among the women with cervical cancer. Cancer prevalence increased with age. The highest prevalence, 9.49%, was found among women older than 50 years, compared with 4.63% for those aged 40 to 50 years, 2.13 % for those aged 30 to 40 years, and 0.61% for those aged 18 to 30 years (Table 1). The 4780 study patients and the 165 controls received cytologic and/or histologic evaluations, and histologic verification was carried out for those with abnormal cytologic results. Of the 4780 study patients, 2919 had cervicitis; 282 had atypical squamous cells of undetermined significance; 683 had grade 1 cervical intraepithelial neoplasia (CIN 1), 389 had CIN 2 lesions, and 236 had CIN 3 lesions; 117 had carcinoma in situ (CIS); and 154 had cervical cancer (CC). A total of 2260 women (47.28%) tested positive for HPV DNA. The most prevalent genotype (19.64%) was HPV-16, followed by HPV-58, HPV-52, HPV-33, HPV-53, and HPV-31. However, only 86 (1.80%) tested positive for HPV-18, which is a very common genotype in Western countries (Table 2). The 6 major HPV types were present in 79.16% of all infected women, and in 89.80% and 84.38%, respectively, of the women with CIS or CC. Moreover, 64.51% of the women with HPV DNA tested positive for HPV-16, HPV-58, and HPV-52 (Table 3). Infections with a single HPV type were detected in 26.05% of the study patients overall and 58.44% of the study patients with CC, compared with 12.73% of the controls. The rates of infection with a single HR type differed significantly between study patients and controls. The patients who had the lowest risk of being infected with a single HPV type were those with cervicitis (OR, 1.74; 95% CI, 1.09– 2.78). Infection with a single HR type was associated with various cervical conditions. The prevalence rates of single infection with
Table 2 Distribution of HPV genotypes among 165 women with no apparent lesions (controls) and 4780 patients with specific lesions.a HPV type
Controls
b
Cervicitis (n = 2919)
ASCUS (n = 282)
CIN (n = 1308)
CIS (n = 117)
CC (n = 154)
Total
High risk 16 58 52 33 31 68 18 39 35 59 51 56 45
18 (10.9) 2 (1.21) 3 (1.82) 1 (0.61) 1 (0.61) 0 1 (0.61) 0 0 1 (0.61) 0 0 0
334 (11.44) 201 (6.89) 155 (5.31) 99 (3.39) 63 (2.16) 45 (1.54) 41 (1.40) 40 (1.37) 36 (1.23) 22 (0.75) 18 (0.62) 16 (0.55) 4 (0.14)
57 (20.2) 33 (11.7) 20 (7.09) 12 (4.26) 8 (2.84) 5 (1.77) 9 (3.19) 5 (1.77) 5 (1.77) 7 (2.49) 3 (1.06) 2 (0.71) 0
392 (30.0) 147 (11.2) 117 (8.94) 97 (7.42) 59 (4.51) 33 (2.52) 25 (1.91) 27 (2.06) 7 (0.54) 15 (1.15) 15 (1.15) 8 (0.61) 7 (0.54)
71 (60.68) 18 (15.38) 3 (2.56) 7 (5.98) 10 (8.55) 2 (1.71) 4 (3.42) 0 1 (0.85) 0 0 0 0
85 (55.19) 12 (7.79) 11 (7.14) 5 (3.25) 5 (3.25) 4 (2.60) 7 (4.55) 4 (2.60) 0 1 (0.65) 1 (0.65) 3 (1.95) 3 (1.95)
939 (19.64) 401 (8.39) 306 (6.40) 220 (4.60) 145 (3.03) 89 (1.86) 86 (1.80) 76 (1.59) 49 (1.03) 45 (0.94) 37 (0.77) 29 (0.61) 14 (0.29)
4 (2.42) 2 (1.21) 1 (0.61)
73 (2.50) 59 (2.02) 39 (1.34)
13 (4.61) 6 (2.13) 7 (2.49)
50 (3.82) 48 (3.67) 28 (2.14)
9 (7.69) 5 (4.27) 6 (5.13)
7 (4.55) 10 (6.49) 6 (3.90)
152 (3.18) 128 (2.68) 86 (1.80)
1 (0.61) 2 (1.21) 0 1 (0.61) 1 (0.61) 36 (21.82)
69 (2.36) 50 (1.71) 18 (0.62) 5 (0.17) 4 (0.14) 1041 (35.66)
5 (1.77) 3 (1.06) 2 (0.71) 1 (0.35) 0 154 (54.61)
25 (1.91) 22 (1.68) 9 (0.69) 4 (0.31) 2 (0.15) 839 (64.14)
1 (0.85) 2 (1.71) 0 1 (0.85) 0 98 (83.76)
0 1 (0.65) 3 (1.95) 1 (0.65) 0 128 (83.12)
100 (2.09) 78 (1.63) 32 (0.67) 12 (0.25) 6 (0.13) 2260 (47.28)c
Intermediate risk 53 CP8304 66 Low risk 6 11 44 42 43 Total a b c
Abbreviations: Please see Table 1; values are given as number (percentage). These are the controls who tested positive for single or multiple infections. Of these 2260 patients, 1529 had a single infection and 731 had a multiple infection.
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Table 3 Major HPV types, alone or in combinations, present in the infected women (study patients and controls combined).a HPV types
Controls (n = 36)b
Cervicitis (n = 1041)
ASCUS (n = 154)
CIN (n = 839)
CIS (n = 98)
CC (n = 128)
Total (N = 2260)
16 16, 16, 16, 16,
18 21 22 25 26
334 622 690 749 791
57 84 90 98 104
392 571 634 663 698
71 (72.45) 79 (80.61) 84 (85.71) 87 (88.78) 88 (89.80)
85 (66.41) 102 (79.69) 102 (79.69) 106 (82.81) 108 (84.38)
939 1458 1600 1703 1789
a b
58, 52 58, 52, 33 58, 52, 33, 53 58, 52, 33, 53, 31
(50.00) (58.33) (61.11) (69.44) (72.22)
(32.08) (59.75) (66.28) (71.95) (75.98)
(37.01) (54.54) (58.44) (63.64) (67.53)
(46.72) (68.05) (75.57) (79.02) (83.19)
(41.55) (64.51) (70.80) (75.35) (79.16)
Abbreviations: Please see Table 1; values are given as number (percentage). These are the controls who tested positive for single or multiple infections.
LR types or types common in China were similar in all subgroups (Table 4). Infection with multiple HPV types was seen in 731 (32.35%) of the 2260 HPV-infected women, study patients and controls combined, or 15.29% of the 4780 study patients. Of these 731 patients, 399 were infected with HPV-16 as well as other HPV types. The rates of multiple infection were significantly higher in each subgroup of patients with a specific cervical disease than in the control group overall. The patients who had the lowest risk of having a multiple infection were those with cervicitis (OR, 3.33; 95% CI, 1.35–8.18). Infection with multiple HPV types may increase the risk of cervical disease (Table 4). Infection with an HR HPV genotype has been shown to be a major risk factor for cervical disease. Single infections with HPV-16 were significantly more prevalent among women with CC than among those with CIN (χ2 = 93.4) or among controls overall (χ2 = 50.8) (P b 0.05). A single infection with HPV-16 was the major risk factor for CC. Although a single infection with HPV-33 was relatively common among patients with CIN, it was rarely detected in those with CC. The prevalence of single infection with HPV-33 was null in the control group. In the study group, it was significantly higher among patients with CIN than among patients with CC (χ2 = 4.37, P b 0.05) (Table 5). 4. Discussion We conducted the present study using the HybriMax GenoArray System, a commercial oligonucleotide chip kit capable of detecting multiple types of HPV in cervical cells. The case-control design of the study allowed us to determine the distribution of HR HPV genotypes among women with cervical disease in northeastern China, as well as the prevalence rates of single and multiple infection with different
HPV types. The associations between HPV infection and grade of cervical lesion were also analyzed. A series of studies has established a relationship between HPV infection and cervical disease, and prevalence rates of HPV infection have been estimated for various populations in southern China [6–8]. We estimated an infection rate of 47.28% among women with cervicitis, preinvasive cervical disease, or invasive cervical cancer in Liaoning province in northeastern China. The most common genotype was HPV-16 in Liaoning province, followed by types 58, 52, 33, 53, and 31. One or more of these 6 genotypes were found in 79.16% of infected patients and accounted for 89.80% and 84.38% of infections, respectively, among women with CIS or CC. The 55.19% rate of HPV-16 infection we report for our CC subgroup is similar to the rate reported by Lo et al. [11]. In our study, HPV-58 was the second most common type, with an infection rate of 7.79% for our CC subgroup. In contrast, Chan et al. [12] reported that one-third of the women with CC in Hong Kong were positive for HPV-58, and similarly high rates have also been reported for Chinese populations living in Shanghai and Taiwan [7,8]. These studies suggest that HPV types 52 and 58 may play a more prominent role in the development of CC in Asia than HPV types 31, 33, and 45, which are more common on other continents [12]. Although HPV-18 has been found to be common in other regions of China and in other countries, we report a prevalence of 1.80% for our study patients and controls combined, and a prevalence of 4.55% for all women with CC, and these rates are lower than those reported for other parts of China. Lo et al. [11] reported a 14.8% rate of HPV-18 infection for Hong Kong women with CC. Another study also showed that HPV types 16 and 18 were the most common types in Indonesia, affecting, respectively, 41.9% and 37.8% of the women with CC [13]. The 35.66% HPV infection rate we are reporting for women with cervicitis is
Table 4 Distribution of single or multiple HPV infections among 165 women with no apparent lesions (controls) and 4780 patients with specific lesions.a HPV types
Controls (n = 165)
Cervicitis (n = 2919)
ASCUS (n = 282)
CIN (n = 1308)
CIS (n = 117)
CC (n = 154)
Total
64 8.28 4 1.13 2 0.557
(54.70) (4.61–14.86) (3.42) (0.30–4.31) (1.71) (0.11–2.92)
90 (58.44) 9.64 (5.51–16.86) 3 (1.95) 0.64 (0.15–2.71) 3 (1.95) 0.64 (0.15–2.71)
1245 (26.05)
28 (23.93) 10.08 (3.76–26.99)
32 (20.78) 8.40 (3.18–22.18)
Single infection High risk types
b
Common in China Low risk types
c
c
21 (12.73) 1.0 5 (3.03) 1.0 5 (3.03) 1.0
592 1.74 98 1.11 76 0.86
(20.28) (1.09–2.78) (3.36) (0.446–2.77) (2.60) (0.34–2.14)
71 (25.18) 2.31 (1.36–3.92) 13 (4.61) 1.55 (0.54–4.42) 5 (1.77) 0.58 (0.17–2.03)
428 3.34 55 1.41 25 0.62
(32.7) (2.08–5.35) (4.20) (0.55–3.56) (1.15) (0.24–1.65)
173 (3.62) 111 (2.32)
Multiple infection Low- and high-risk typesd a
5 (3.03) 1.0
275 (9.42) 3.33 (1.35–8.18)
65 (23.05) 9.59 (3.77–24.35)
331 (25.31) 10.84 (4.41–26.63)
731 (15.29)e
Abbreviations: Please see Table 1; values are given as number (percentage) and odds ratio (OR) and 95% confidence interval (CI). When the ORs for having a single HPV infection in the control group were compared with the corresponding ORs in each of these subgroups of patients, the differences were significantly different (χ2 = 5.63, P b 0.05 for the smallest difference). c When the ORs for having a single infection in the control group were compared with the corresponding ORs in each of these subgroups of patients, the differences were not significantly different (P N 0.05 in all cases). d When the ORs for having a multiple HPV infection in the control group were compared with the corresponding ORs in each of these subgroups of patients, the differences were significantly different (χ2 = 7.73, P b 0.05 for the smallest difference). e Among the 731 patients with multiple infections, 399 (54.58%) were infected by HPV-16 as well as other types. b
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Table 5 Distribution of 2 single HPV infections among controls and patients with CIN and CC.a HPV type
Controls (n = 165)
CIN (n = 1308)
CC (n = 154)
HPV-16 HPV-33
15 (9.09) 0
176 (13.46) 45 (3.44)c
68 (44.16)b 0
Abbreviations: CIN, cervical intraepithelial neoplasia; CC, cervical cancer. a Values are given as number (percentage). b The difference between the percentage of HPV-16 single infection in patients in the cervical cancer group and those in the CIN group or healthy women was statistically significant (χ2 = 93.4 and χ2 = 50.8, respectively, P b 0.05 for both). c The difference between the percentage of HPV-33 single infection in patients in the CIN group and those in the cervical cancer group or healthy women was statistically significant (χ2 = 4.37 for both, P b 0.05).
similar to the 33.1% rate (46 of 139 women) reported by Wu et al. [6] for Guangdong province in China. In our study, however, the rates of HPV infection for women with CIN 1, CIN 2, and CIN 3 (50.66%, 77.37% and 81.36%, respectively) are lower than those reported by these authors. Of the 154 study patients with CC, 128 (83.12%) were infected with HPV, and the difference with the controls was significant. This result is similar to the 89.9% (427 of 475 patients) reported for Guangdong province [6] and the 83% (88 of 106 patients) reported for southern China [7]. In this study, we analyzed infection with single HR HPV types in women with various cervical conditions, and the rates increased with lesion severity. This finding was consistent with previous findings [14]. Regarding infection with single LR HPV types or single HR types common in China, however, our findings did not match previous findings. The effect of multiple HPV infection on CC pathogenesis is unclear. Herrero et al. [15] found that women infected with HPV-16 alone were at similar or higher risk for CC than women infected with HPV-16 and another HPV type, but Lee et al. [16] reported an association between infection with multiple HPV types and increased risk of CC. Of the 4780 patients in the present study, 731 (15.29%) were infected with multiple HPV types; and of the 2260 women found to be infected with HPV in the study and control groups combined, 731 (32.35%) were infected with multiple HPV types. This is higher than in other studies, but the reported prevalence of multiple HPV infections varies widely, from 3.9% in Thailand [17], to 12.9% among women with squamous tumors in Peru [18], to 19.3% in Paraguay [19], to 32.2% in Korea [20], to 32% in Costa Rica [15], and to 34.3% in Mozambique [21]. These differences could be due to different rates of multiple infection in the various populations or to differences in the detection methods [22]. Of the 731 cases of multiple infections in our study, 399 involved HPV-16 plus other types. The difference between the rates of multiple HPV infection in the study patient and control groups was statistically significant. Infection with multiple types of HPV can increase the risk of cervical disease. It has been demonstrated that HPV infection is the main risk factor for the development of CC, and that HPV-16 is the genotype most frequently found in women with multiple HPV infections [23]. In our study, a single infection with HPV-16 was significantly more prevalent among women with CC than among women with CIN or among controls. Infection with HPV-16 alone can cause cervical cancer, and it is the main risk factor of CC in northern China. One of our findings is worth stressing. Whereas none of the 154 women with CC was infected with HPV-33, 45 of the 1308 women with CIN were. A single infection with HPV-33 therefore seemed more likely to cause CIN than CC in our population, and it will be worthwhile to test this observation in future studies. Information regarding HPV types found in given populations is very important for vaccine design. Based on our findings, and assuming that the efficacy of HPV vaccines containing HPV-16 is close to 100% [24], we could assume that about 55.19% of cases of CC would be prevented by using these vaccines in northeastern China. Our study provides basic prevalence data for HPV types in Liaoning province that can be
used to increase vaccine efficacy in that province. The prevalence of multiple infection with HPV-16, HPV-58, and HPV-52 was 79.69% for patients with CC. If the HPV vaccines also contained more HPV types, such as HPV-33, HPV-53, and HPV-31, the preventive efficacy should reach 84.38% for CC and 79.16% for any sort of precancerous lesion. An HPV vaccine for use in northeastern China should be designed against these 6 major HPV types first. Our findings further emphasize the importance of a vaccination program in northeastern China aimed at immunizing women before they become infected with HPV. Acknowledgment This work was supported by the National Natural Science Foundation of China (grant 30672248), the National Natural Science Foundation of China (grant 30770109), and the Project of Doctor Foundation of Liaoning Province in China (grant 20061039). Conflict of interest The authors declare that they have no conflict of interest. References [1] Van Tine BA, Kappes JC, Banerjee NS, Knops J, Lai L, Steenbergen RD, et al. Clonal selection for transcriptionally active viral oncogenes during progression to cancer. J Virol 2004;78(20):11172–86. [2] Schlecht NF, Kulaga S, Robitaille J, Ferreira S, Santos M, Miyamura RA, et al. Persistent human papillomavirus infection as a predictor of cervical intraepithelial neoplasia. JAMA 2001;286(24):3106–14. [3] Muñoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348(6):518–27. [4] zur Hausen H. Papillomavirus infections: a major cause of human cancers. Biochim Biophys Acta 1996;1288(2):55–78. [5] Alam S, Bromberg-White J, McLaughlin-Drubin M, Sen E, Bodily JM, Meyers C. Activity and therapeutic potential of ORI-1001 antisense oligonucleotide on human papillomavirus replication utilizing a model of dysplastic human epithelium. Anticancer Res 2005;25(2A):765–77. [6] Wu Y, Chen Y, Li L, Yu G, Zhang Y, He Y. Associations of high-risk HPV types and viral load with cervical cancer in China. J Clin Virol 2006;35(3):264–9. [7] Liu J, Rose B, Huang X, Liao G, Carter J, Wu X, et al. Comparative analysis of characteristics of women with cervical cancer in highversus low-incidence regions. Gynecol Oncol 2004;94(3):803–10. [8] Lai CH, Chao A, Chang CJ, Chao FY, Huang HJ, Hsueh S, et al. Host and viral factors in relation to clearance of human papillomavirus infection: a cohort study in Taiwan. Int J Cancer 2008;123(7):1685–92. [9] Grisaru D, Avidor B, Niv J, Marmor S, Almog B, Leibowitz C, et al. Pilot study of prevalence of high-risk human papillomavirus genotypes in Israeli Jewish women referred for colposcopic examination. J Clin Microbiol 2008;46(5):1602–4. [10] Carozzi F, Bisanzi S, Sani C, Zappa M, Cecchini S, Ciatto S, et al. Agreement between the Amplicor HPV test and the Hybrid Capture 2 assay in the detection of high-risk human papillomavirus and biopsy-confirmed high-grade cervical disease. J Clin Microbiol 2007;45(2):364–9. [11] Lo KW, Wong YF, Chan MK, Li JC, Poon JS, Wang VW, et al. Prevalence of human papillomavirus in cervical cancer: a multicenter study in China. Int J Cancer 2002;100(3):327–31. [12] Chan PK, Lam CW, Cheung TH, Li WW, Lo KW, Chan MY, et al. Association of human papillomavirus type 58 variant with the risk of cervical cancer. J Natl Cancer Inst 2002;94(16):1249–53. [13] Schellekens MC, Dijkman A, Aziz MF, Siregar B, Cornain S, Kolkman-Uljee S, et al. Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. Gynecol Oncol 2004;93(1):49–53. [14] Sun CA, Lai HC, Chang CC, Neih S, Yu CP, Chu TY. The significance of human papillomavirus viral load in prediction of histologic severity and size of squamous intraepithelial lesions of uterine cervix. Gynecol Oncol 2001;83(1):95–9. [15] Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, Morales J, et al. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 2000;92(6):464–74. [16] Lee SA, Kang D, Seo SS, Jeong JK, Yoo KY, Jeon YT, et al. Multiple HPV infection in cervical cancer screened by HPVDNAChip. Cancer Lett 2003;198(2):187–92. [17] Chichareon S, Herrero R, Muñoz N, Bosch FX, Jacobs MV, Deacon J, et al. Risk factors for cervical cancer in Thailand: a case-control study. J Natl Cancer Inst 1998;90(1): 50–7. [18] Santos C, Muñoz N, Klug S, Almonte M, Guerrero I, Alvarez M, et al. HPV types and cofactors causing cervical cancer in Peru. Br J Cancer 2001;85(7):966–71. [19] Rolón PA, Smith JS, Muñoz N, Klug SJ, Herrero R, Bosch X, et al. Human papillomavirus infection and invasive cervical cancer in Paraguay. Int J Cancer 2000;85(4):486–91. [20] Huang HJ, Huang SL, Lin CY, Lin RW, Chao FY, Chen MY, et al. Human papillomavirus genotyping by a polymerase chain reaction-based genechip method in cervical
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