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Prevalence of high-risk HPV and associated risk factors in cases of cervical carcinoma in Tamil Nadu, India
International Journal of Gynecology and Obstetrics 119 (2012) 253–256
Contents lists available at SciVerse ScienceDirect
International Journal of Gynecology and Obstetrics journal homepage: www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Prevalence of high-risk HPV and associated risk factors in cases of cervical carcinoma in Tamil Nadu, India Krishnakumar Vinodhini a, Santhanam Shanmughapriya a, Sumathy Sanmugham b, Ganesan Senthikumar c, Bhudev C. Das d, Kalimuthusamy Natarajaseenivasan a,⁎ a
Medical Microbiology Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirapalli, India KAPV Medical College, Tiruchirapalli, India Dr G. Vishwanthan Cancer Institute, Tiruchirapalli, India d Dr B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India b c
a r t i c l e
i n f o
Article history: Received 2 November 2011 Received in revised form 22 June 2012 Accepted 22 August 2012 Keywords: HPV India Invasive cervical cancer Prevalence Screening
a b s t r a c t Objective: To assess the prevalence of HPV infection among women with cervical cancer in Tiruchirapalli, Tamil Nadu, India. Methods: A case–control study was conducted with 246 women with cervical cancer and 257 control participants aged between 20 and 70 years. The presence of HPV DNA was determined using the MY09/11 PCR protocol, the GP5 +/6+ PCR protocol, and type-specific PCR-based assays. Results: The overall HPV prevalence was 54.9% and HPV 16 was the most prevalent type. The women the most vulnerable for HPV infection were those aged 41 to 60 years. The major contributing risk factors for cervical cancer were having had 3 or more pregnancies (OR 19.2; 95% CI, 10.78–10.16); harboring high-risk HPV DNA (OR 15.3; 95% CI, 9.81–23.8); being a manual worker (OR 14.9; 95% CI, 9.62–23.08); being illiterate (OR 8.28; 95% CI, 5.51–12.43); and having never been screened for cervical dysplasia (OR 6.70; 95% CI, 10.78–34.44). Conclusion: The present report on the overall and type-specific prevalence rates and risk burden of HPV infection in Tamil Nadu confirms that screening and vaccination programs are urgently needed in this state to overcome the burden of HPV-associated cervical cancer. © 2012 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cervical cancer is the most common cancer among Indian women and the second most common cancer worldwide, and is therefore a serious global public health concern [1]. Of the 493 000 new diagnoses per year, 80% are reported from low-resource countries and 25% from India alone [2]. Infection with certain genotypes of HPV has been linked with cervical cancer development [3]. The lifetime risk for sexually active women to be infected with HPV is 70%; and whereas most HPV infections are transient, persistent cervical infection with a high-risk genotype facilitates the development of cervical intraepithelial neoplasia [4]. The high-risk types of HPV (HR-HPV) are HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, but HPV 16 and 18 are among the most likely to cause cancer [5,6]. Vaccines were developed to combat HPV infection [7]; and since in each region a vaccine targeting the most prevalent HPV types would have the greatest impact, assessing the presence of HR-HPV DNA during primary screening is of prime importance. In countries such as India, however, there is an urgent need to improve screening programs to reduce the cancer burden. The aim of the present case–control ⁎ Corresponding author at: Medical Microbiology Laboratory, Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirapalli–620 024, India. Tel.: +91 4312407082; fax: +91 94312407045. E-mail address: [email protected] (K. Natarajaseenivasan).
study was to determine the prevalent HPV types among women with invasive cervical cancer (ICC) in the Tiruchirapalli district of the state of Tamil Nadu, India. 2. Materials and methods The study was approved by the Institutional ethics committee of Bharathidasan University (DM/07/101/373) and carried out at the Obstetrics and Gynecology Department of Annal Gandhi Memorial– Government Hospital (AGMGH), a primary and oncology care hospital located in Tiruchirapalli, Tamil Nadu, India. The medical records of AGMGH were reviewed and 246 women newly diagnosed with ICC were recruited between May 2, 2009, and May 30, 2011. The inclusion and exclusion criteria for this group were those proposed by Gargiulo et al. [8]: no previous cancer treatment, no history of other gynecologic cancer, no physical or mental impairment, and histologic confirmation of ICC diagnosis. The control group consisted of 257 women seen at the same hospital during the same period for routine gynecologic care. The criteria for inclusion in the control group were the following: no abnormal cytologic findings, no history of cervical conization or hysterectomy, no physical or mental impairment, and not being pregnant. All recruited women received detailed information regarding the objective of the study and gave written consent to participate. Their
0020-7292/$ – see front matter © 2012 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijgo.2012.06.019
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K. Vinodhini et al. / International Journal of Gynecology and Obstetrics 119 (2012) 253–256
sociodemographic characteristics were then recorded and their reproductive and contraceptive histories taken. For HPV detection, the participants in the case group underwent a punch biopsy whereas cervical scrapes were collected from the controls by means of wooden spatulas. The biopsy specimens were frozen immediately at − 20 °C. The spatulas with the cervical scrapes were placed in an ice-cold phosphate-buffered saline solution and transported to the laboratory on ice. The cell suspension was centrifuged at 3000 g for 10 minutes at 4 °C, the supernatant was discarded, and the cell pellets were stored at − 20 °C until processing. For DNA extraction, the standard SDS-proteinase K-phenol chloroform method [9] was used for the biopsy samples and the boiling method [10,11] was used for the cervical scrapes. The quality of the extracted DNA was verified by amplification of a 268-bp region of the human β-globin gene using PCO4 and GH20 primers [12], and HPV DNA was then first amplified using the universal MY09/11 polymerase chain reaction (PCR) protocol [13,14]. The samples negative for HPV DNA by the MY09/11 PCR protocol were further assessed via the GP5 +/6 + PCR protocol [15]. Moreover, type-specific primers were used for HPV types 16, 18, 6, and 11 and the consensus primers pU-1 M/pU-2R and pU-31B were used for HPV types 31, 33, 52b, and 58. The amplified PCR products were visualized on 1% agarose gel stained with ethidium bromide and documented using a gel documentation system (Gel Doc XR+; BioRad, Hercules, CA, USA). The data were coded and analyzed using SPSS version 17.0 (IBM, Armonk, NY, USA). Unconditional logistic regression was performed and odds ratio (OR) and corresponding 95% confidence intervals (CIs) were calculated. P b 0.05 was considered significant.
3. Results The mean ± SD age was 49.6 ± 2.34 years for the study patients and 47.2 ± 2.44 years for the controls. Nearly 56% of the participants in the case group (i.e. those with ICC) were aged 41 to 60 years, 23.2% were aged 21 to 40 years, and 21% were older than 60 years. Table 1 summarizes the socioeconomic, behavioral, and reproductive characteristics of the participants in both groups. Being infected with a HR-HPV type (OR 15.3; 95% CI, 9.81–23.8), being illiterate (OR 8.28; 95% CI, 5.51–12.43), being employed as a manual worker (OR 14.9; 95% CI 9.62–23.08), having had 3 or more pregnancies (OR 19.2; 95% CI, 10.78–10.16), never having been screened for dysplasia (OR 6.70; 95% CI, 10.78–34.44), and being 15 years or younger at first coitus (OR 4.21; 95% CI, 3.82–8.82) were significantly associated with the presence of cervical cancer. Table 2 summarizes the relation between HPV infection and the presence of cervical cancer. Infection with HPV of any type increased the risk of developing cervical cancer 10-fold (OR 10.66; 95% CI, 16.6–15.34), and infection with HPV 16 increased the risk even more (OR 11.18; 95% CI, 6.06–20.61). Samples from 9 of the 503 participants were insufficient for DNA extraction. Of the 494 remaining samples, 271 were positive for HPV DNA, for an overall prevalence of 54.9% (Fig. 1). Although the case group had the highest prevalence of HPV infection (81.4%), the prevalence was 30.4% in the control group where no women had abnormal cytologic findings. There was HPV 16 DNA in the cervical samples of 159 (82.3%) women in the case group and 23 controls (29.4%); HPV 18 DNA in the samples of 16 (8.29%) women in the case group and 17 controls (21.7%); and HPV 6 or HPV 11 DNA—which are both low-risk types responsible for genital warts—in the samples of 13 (6.73%) women in the case group and 28 controls (35.8%). No women in the control group but 16 women (8.2%) in the case group were infected with HPV 16 and 1 or more other types. In the case group, the prevalence of HPV infection was highest (63.7%) among the women aged 41 to 60 years; it was 19.6% among those aged 60 to 80 years; and it was 16.5% among those aged 21 to 40 years. The women aged 41 to 60 years were the most likely to harbor HPV DNA.
Table 1 Cervical cancer associations with behavioral, sociodemographic, sexual, and reproductive history.a Characteristic
Cases (n = 237)
HPV infection HPV16/18 175 (73.8) HPV6/11 13 (5.4) Other 7 (2.9) Education None 163 (68.7) Primary 54 (22.7) Some secondary 16 (6.7) High school graduate 4 (1.6) Annual income, Indian rupees b20 000 96 (40.5) 21 000–40 000 81 (34.1) >40 000 60 (25.3) Occupation Manual worker 191 (80.5) Housewife 46 (19.4) Age at first coitus, y ≤15 107 (45.1) 16–20 81 (34.1) 21–25 15 (6.3) >25 34 (14.3) Age at menarche, y b12 21 (8.8) 13–14 148 (62.4) ≥15 68 (28.6) No. of pregnancies 0 11 (4.6) 1–2 58 (24.4) 2–3 39 (16.4) ≥3 129 (54.4) Marital status Cohabiting 182 (76.7) Separated 48 (20.2) Widowed 7 (2.9) Frequency of sexual intercourseb Rarely 42 (17.7) ≥3 per wk 136 (57.3) 1 per wk 40 (16.8) 2 per mo 19 (8.0) No. of sexual partners 0–1 204 (86.0) ≥3 28 (13.9) No. of abortions 0 101 (42.6) 1–2 34 (14.3) ≥3 102 (43.0) Cervical screening Never 148 (62.4) 1–2 53 (22.3) ≥3 36 (15.1)
Controls (n = 257) 40 (15.5) 28 (10.8) 10 (3.8) 54 85 109 13
(21.0) (33.0) (42.4) (5.0)
OR (95% CI)
P value
15.30 (9.81–23.80) 0.40 (0.23–0.93) 0.70 (0.28–2.00)
b0.001 0.53 0.40
8.28 0.597 0.27 0.32
(5.51–12.43) (0.40–0.89) (0.15–0.49) (0.10–1.00)
0.004 0.45 0.60 0.57
137 (53.3) 62 (24.1) 58 (22.5)
0.59 (0.42–0.85) 1.63 (1.10–2.41) 1.10 (0.77–1.76)
0.44 0.20 0.29
46 (17.8) 191 (74.3)
14.9 (9.62–23.08) 0.06 (0.04–0.10)
0.001 0.81
42 126 38 51
4.21 0.33 0.67 0.38
(3.82–8.82) (0.22–0.48) (0.42–1.08) (0.20–0.72)
0.04 0.57 0.41 0.54
46 (17.8) 73 (28.4) 138 (53.6)
0.44 (0.25–0.77) 4.19 (2.87–6.11) 0.34 (0.23–0.50)
0.51 0.04 0.56
13 105 64 15
0.91 0.49 0.59 19.2
(16.3) (49.0) (14.7) (19.8)
(5.0) (40.8) (24.9) (5.8)
149 (57.9) 87 (33.8) 61 (23.7) 77 43 53 84
(29.9) (16.7) (20.6) (32.6)
(0.40–2.08) (0.31–0.69) (0.38–0.92) (10.78–34.44)
0.34 0.48 0.44 b0.001
2.39 (1.62–3.54) 0.49 (0.32–0.74) 0.09 (0.04–0.21)
0.12 0.48 0.76
0.82 6.70 1.74 0.43
0.36 0.01 0.19 0.51
(0.55–1.22) (4.41–10.16) (1.16–2.6) (0.24–0.76)
238 (92.6) 19 (7.3)
0.58 (0.32–1.09) 1.67 (0.91–3.09)
0.45 0.20
109 (42.4) 111 (43.1) 37 (14.3)
1.00 (0.70–1.44) 0.22 (0.14–0.34) 4.49 (2.91–6.92)
0.32 0.65 0.03
32 (12.4) 86 (33.4) 139 (54.0)
11.60 (7.42–18.41) 0.80 (0.60–1.29) 0.15 (0.09–0.23)
0.007 0.37 0.70
Abbreviations: CI, confidence interval; OR, odds ratio. a Values are given as number (percentage) unless otherwise indicated.
4. Discussion Invasive cervical cancer was found to be the most frequent cancer among women, and infection with HPV to be the major cause for its development [16]. Most studies have shown HPV prevalence to be Table 2 HPV infection and its association with cervical cancer. HPV Infection
Cases(n = 237) Controls(n = 257) OR (95% CI)
HPV infection, any type Positive 193 Negative 44 HPV 16 159 HPV 18 16 HPV 16-related types 21
78 179 23 17 0
10.66 (6.6–15.34) 0.01 (0.06–0.15) 11.18 (6.06–20.61) 0.32 (0.15–0.68) NA
Abbreviations: CI, confidence interval; NA, not applicable; OR, odds ratio.
K. Vinodhini et al. / International Journal of Gynecology and Obstetrics 119 (2012) 253–256
Fig. 1. PCR-based detection of HPV infection. (A) Polymerase chain reaction (PCR) amplification of the HPV L1 capsid protein gene using MY09 and MY11 primers: lane M, 1-kb DNA marker showing 7 PCR amplicons of 450 bp from cervical carcinomas. (B) Nested PCR amplification of the HPV L1 gene using the degenerate primer pair GP5+/6+: lane M, 100-bp DNA marker showing 7 PCR amplicons of 150 bp. (C) PCR amplification of HPV 16 using type-specific primers: lane M, 1-kb DNA marker showing 5 PCR amplicons of 216 bp. (D) PCR amplification of HPV 18 using type-specific primers: lane M, 100-bp DNA marker showing 7 PCR amplicons of 118 bp. (E) PCR amplification of HPV 6 and HPV 11 with consensus primers: lane M, 100-bp DNA marker showing 7 PCR amplicons of 68 bp.
255
The prevalence of HPV infection increased from age group to age group until the 41- to 60-year-old group, after which it declined. A second peak concerning women older than 65 years has been reported elsewhere [6,21] but was not observed in the present study. There was therefore no reactivation of latent viruses among the study population. The same decrease in HPV prevalence in populations of increasing age has been reported in Asia [6]. Many epidemiologic studies have reported a large number of sexual partners as a major risk factor for the development of cervical cancer [22]. However, owing to the prevailing customs, a large number of partners is very rare in India, and this factor was not found to be significant. In addition to behavioral and sexual factors, infection with HR-HPV types was an important factor for the development of cervical cancer. The participants had no adequate knowledge about cervical cancer and ways of preventing it. There are no established screening programs for cervical dysplasia in Tamil Nadu, and there is certainly an urgent need to implement one in Tiruchirapalli and run it with sufficient infrastructure, staff, and other needed resources—and complement it with HPV testing as a potential alternative to cytologic screening and a preventive measure for cervical cancer. Acknowledgments The study was supported by the Indian Council of Medical Research (No. 5/13/88/06–NCD–III). Conflict of interest The authors have no conflicts of interest.
associated with several factors, which include societal economic development as well as the educational level and sexual behavior of infected persons. The prevalence of HPV infection was estimated to be 15.5% in low-income countries and 10.0% in high-income countries [17]. The present case–control study determined the distribution of HR-HPV genotypes among women with cervical cancer who live in the Tiruchirapalli district of the state of Tamil Nadu in southern India. Whereas the prevalence of HPV infection was found to be 54.9% for cases and controls combined, 82.3% of the cases, defined as women having ICC, were found to be harboring HPV DNA—a finding which was consistent with GLOBOCAN data (http://globocan.iarc. fr/factsheet.asp). Worldwide, half of all HPV infections are caused by HPV 16, 18, 31, 58, and 52. Various studies have reported HPV 16 as the most prevalent type except in eastern Africa, Japan, and Taiwan where the most common is HPV 52 [6]. In the present in study, the most common was HPV 16, for a prevalence of 29.4%. The overall prevalence of HPV 16 reported for the study population is slightly lower than the lower end of the 34.7% to 59.6% range reported in other studies [18]. In the control group, the HPV prevalence was 30.4% and the types were mostly the low-risk HPV 6 and HPV 11. The present results do not differ much from those previously reported for Tamil Nadu [19,20] and, thus, substantiate the hypothesis that HPV infection alone is not responsible for the progression of cervical cancer. A persistent infection, with integration of HPV genome into the chromosomal DNA of cervical epithelial cells, along with alterations of human oncogenes and tumor suppressor genes in the affected cells, and the impairment of immune mechanisms combine to enable the development of cervical cancer. Thus, HPV infection in women with normal cytologic findings can be attributed to the presence of genital warts, the latter sustaining the persistence of HPV infection. Infection with more than 1 genotype was detected in 8.2% of the women infected with HPV. Several studies have noted that patients could be co-infected with HPV of different genotypes. In the present study, some participants were co-infected with HPV 16 and 1 or more of other genotypes.
References [1] Gheit T, Vaccarella S, Schmitt M, Pawlita M, Franceschi S, Sankaranarayanan R, et al. Prevalence of human papillomavirus types in cervical and oral cancers in central India. Vaccine 2009;27(5):636-9. [2] Bhatla N, Lal N, Bao YP, Ng T, Qiao YL. A meta–analysis of human papillomavirus type–distribution in women from South Asia: implications for vaccination. Vaccine 2008;26(23):2811-7. [3] 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. [4] 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. [5] Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348(6):518-27. [6] zur Hausen H. Papillomavirus infections––a major cause of human cancers. Biochim Biophys Acta 1996;1288(2):F55-78. [7] 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. [8] Gargiulo F, De Francesco MA, Schreiber C, Ciravolo G, Salinaro F, Valloncini B, et al. Prevalence and distribution of single and multiple HPV infections in cytologically abnormal cervical samples from Italian women. Virus Res 2007;125(2):176-82. [9] Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim–van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28(3):495-503. [10] Gopalkrishna V, Srivastava AN, Hedau S, Sharma JK, Das BC. Detection of human papillomavirus DNA sequences in cancer of the urinary bladder by in situ hybridisation and polymerase chain reaction. Genitourin Med 1995;71(4):231-3. [11] Das BC, Sharma JK, Gopalakrishna V, Luthra UK. Analysis by polymerase chain reaction of the physical state of human papillomavirus type 16 DNA in cervical preneoplastic and neoplastic lesions. J Gen Virol 1992;73(Pt 9):2327-36. [12] Hildesheim A, Schiffman MH, Gravitt PE, Glass AG, Greer CE, Zhang T, et al. Persistence of type–specific human papillomavirus infection among cytologically normal women. J Infect Dis 1994;169(2):235-40. [13] Bauer HM, Greer CE, Manos MM. Determination of Genital HPV Infection using Consensus PCR. In: Herrington CS, McGee JO, editors. Diagnostic Molecular Pathology: A Practical Approach. Oxford: IRL Press; 1992. p. 131-52. [14] Bauer HM, Hildesheim A, Schiffman MH, Glass AG, Rush BB, Scott DR, et al. Determinants of genital human papillomavirus infection in low–risk women in Portland, Oregon. Sex Transm Dis 1993;20(5):274-8.
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[15] de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ. The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 1995;76(Pt 4):1057-62. [16] González Sánchez JL, Chávez Brambila J, Hernández Hernández DM, Martínez Sánchez S, García Carranca A. High and low risk human papilloma virus infection in women with CIN. Differential characteristics. Ginecol Obstet Mex 2002;70:11-6. [17] Beutner KR, Tyring S. Human papillomavirus and human disease. Am J Med 1997;102(5A):9–15. [18] Bosch FX, Lorincz A, Muñoz N, Meijer CJ, Shah KV. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002;55(4):244-65. [19] Sowjanya AP, Jain M, Poli UR, Padma S, Das M, Shah KV, et al. Prevalence and distribution of high–risk human papilloma virus (HPV) types in invasive squamous
cell carcinoma of the cervix and in normal women in Andhra Pradesh, India. BMC Infect Dis 2005;5:116. [20] Franceschi S, Rajkumar T, Vaccarella S, Gajalakshmi V, Sharmila A, Snijders PJ, et al. Human papillomavirus and risk factors for cervical cancer in Chennai, India: a case–control study. Int J Cancer 2003;107(1):127-33. [21] 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. [22] Currin LG, Jack RH, Linklater KM, Mak V, Møller H, Davies EA. Inequalities in the incidence of cervical cancer in South East England 2001–2005: an investigation of population risk factors. BMC Public Health 2009;9:62.
Contents lists available at SciVerse ScienceDirect
International Journal of Gynecology and Obstetrics journal homepage: www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Prevalence of high-risk HPV and associated risk factors in cases of cervical carcinoma in Tamil Nadu, India Krishnakumar Vinodhini a, Santhanam Shanmughapriya a, Sumathy Sanmugham b, Ganesan Senthikumar c, Bhudev C. Das d, Kalimuthusamy Natarajaseenivasan a,⁎ a
Medical Microbiology Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirapalli, India KAPV Medical College, Tiruchirapalli, India Dr G. Vishwanthan Cancer Institute, Tiruchirapalli, India d Dr B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India b c
a r t i c l e
i n f o
Article history: Received 2 November 2011 Received in revised form 22 June 2012 Accepted 22 August 2012 Keywords: HPV India Invasive cervical cancer Prevalence Screening
a b s t r a c t Objective: To assess the prevalence of HPV infection among women with cervical cancer in Tiruchirapalli, Tamil Nadu, India. Methods: A case–control study was conducted with 246 women with cervical cancer and 257 control participants aged between 20 and 70 years. The presence of HPV DNA was determined using the MY09/11 PCR protocol, the GP5 +/6+ PCR protocol, and type-specific PCR-based assays. Results: The overall HPV prevalence was 54.9% and HPV 16 was the most prevalent type. The women the most vulnerable for HPV infection were those aged 41 to 60 years. The major contributing risk factors for cervical cancer were having had 3 or more pregnancies (OR 19.2; 95% CI, 10.78–10.16); harboring high-risk HPV DNA (OR 15.3; 95% CI, 9.81–23.8); being a manual worker (OR 14.9; 95% CI, 9.62–23.08); being illiterate (OR 8.28; 95% CI, 5.51–12.43); and having never been screened for cervical dysplasia (OR 6.70; 95% CI, 10.78–34.44). Conclusion: The present report on the overall and type-specific prevalence rates and risk burden of HPV infection in Tamil Nadu confirms that screening and vaccination programs are urgently needed in this state to overcome the burden of HPV-associated cervical cancer. © 2012 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cervical cancer is the most common cancer among Indian women and the second most common cancer worldwide, and is therefore a serious global public health concern [1]. Of the 493 000 new diagnoses per year, 80% are reported from low-resource countries and 25% from India alone [2]. Infection with certain genotypes of HPV has been linked with cervical cancer development [3]. The lifetime risk for sexually active women to be infected with HPV is 70%; and whereas most HPV infections are transient, persistent cervical infection with a high-risk genotype facilitates the development of cervical intraepithelial neoplasia [4]. The high-risk types of HPV (HR-HPV) are HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, but HPV 16 and 18 are among the most likely to cause cancer [5,6]. Vaccines were developed to combat HPV infection [7]; and since in each region a vaccine targeting the most prevalent HPV types would have the greatest impact, assessing the presence of HR-HPV DNA during primary screening is of prime importance. In countries such as India, however, there is an urgent need to improve screening programs to reduce the cancer burden. The aim of the present case–control ⁎ Corresponding author at: Medical Microbiology Laboratory, Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirapalli–620 024, India. Tel.: +91 4312407082; fax: +91 94312407045. E-mail address: [email protected] (K. Natarajaseenivasan).
study was to determine the prevalent HPV types among women with invasive cervical cancer (ICC) in the Tiruchirapalli district of the state of Tamil Nadu, India. 2. Materials and methods The study was approved by the Institutional ethics committee of Bharathidasan University (DM/07/101/373) and carried out at the Obstetrics and Gynecology Department of Annal Gandhi Memorial– Government Hospital (AGMGH), a primary and oncology care hospital located in Tiruchirapalli, Tamil Nadu, India. The medical records of AGMGH were reviewed and 246 women newly diagnosed with ICC were recruited between May 2, 2009, and May 30, 2011. The inclusion and exclusion criteria for this group were those proposed by Gargiulo et al. [8]: no previous cancer treatment, no history of other gynecologic cancer, no physical or mental impairment, and histologic confirmation of ICC diagnosis. The control group consisted of 257 women seen at the same hospital during the same period for routine gynecologic care. The criteria for inclusion in the control group were the following: no abnormal cytologic findings, no history of cervical conization or hysterectomy, no physical or mental impairment, and not being pregnant. All recruited women received detailed information regarding the objective of the study and gave written consent to participate. Their
0020-7292/$ – see front matter © 2012 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijgo.2012.06.019
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sociodemographic characteristics were then recorded and their reproductive and contraceptive histories taken. For HPV detection, the participants in the case group underwent a punch biopsy whereas cervical scrapes were collected from the controls by means of wooden spatulas. The biopsy specimens were frozen immediately at − 20 °C. The spatulas with the cervical scrapes were placed in an ice-cold phosphate-buffered saline solution and transported to the laboratory on ice. The cell suspension was centrifuged at 3000 g for 10 minutes at 4 °C, the supernatant was discarded, and the cell pellets were stored at − 20 °C until processing. For DNA extraction, the standard SDS-proteinase K-phenol chloroform method [9] was used for the biopsy samples and the boiling method [10,11] was used for the cervical scrapes. The quality of the extracted DNA was verified by amplification of a 268-bp region of the human β-globin gene using PCO4 and GH20 primers [12], and HPV DNA was then first amplified using the universal MY09/11 polymerase chain reaction (PCR) protocol [13,14]. The samples negative for HPV DNA by the MY09/11 PCR protocol were further assessed via the GP5 +/6 + PCR protocol [15]. Moreover, type-specific primers were used for HPV types 16, 18, 6, and 11 and the consensus primers pU-1 M/pU-2R and pU-31B were used for HPV types 31, 33, 52b, and 58. The amplified PCR products were visualized on 1% agarose gel stained with ethidium bromide and documented using a gel documentation system (Gel Doc XR+; BioRad, Hercules, CA, USA). The data were coded and analyzed using SPSS version 17.0 (IBM, Armonk, NY, USA). Unconditional logistic regression was performed and odds ratio (OR) and corresponding 95% confidence intervals (CIs) were calculated. P b 0.05 was considered significant.
3. Results The mean ± SD age was 49.6 ± 2.34 years for the study patients and 47.2 ± 2.44 years for the controls. Nearly 56% of the participants in the case group (i.e. those with ICC) were aged 41 to 60 years, 23.2% were aged 21 to 40 years, and 21% were older than 60 years. Table 1 summarizes the socioeconomic, behavioral, and reproductive characteristics of the participants in both groups. Being infected with a HR-HPV type (OR 15.3; 95% CI, 9.81–23.8), being illiterate (OR 8.28; 95% CI, 5.51–12.43), being employed as a manual worker (OR 14.9; 95% CI 9.62–23.08), having had 3 or more pregnancies (OR 19.2; 95% CI, 10.78–10.16), never having been screened for dysplasia (OR 6.70; 95% CI, 10.78–34.44), and being 15 years or younger at first coitus (OR 4.21; 95% CI, 3.82–8.82) were significantly associated with the presence of cervical cancer. Table 2 summarizes the relation between HPV infection and the presence of cervical cancer. Infection with HPV of any type increased the risk of developing cervical cancer 10-fold (OR 10.66; 95% CI, 16.6–15.34), and infection with HPV 16 increased the risk even more (OR 11.18; 95% CI, 6.06–20.61). Samples from 9 of the 503 participants were insufficient for DNA extraction. Of the 494 remaining samples, 271 were positive for HPV DNA, for an overall prevalence of 54.9% (Fig. 1). Although the case group had the highest prevalence of HPV infection (81.4%), the prevalence was 30.4% in the control group where no women had abnormal cytologic findings. There was HPV 16 DNA in the cervical samples of 159 (82.3%) women in the case group and 23 controls (29.4%); HPV 18 DNA in the samples of 16 (8.29%) women in the case group and 17 controls (21.7%); and HPV 6 or HPV 11 DNA—which are both low-risk types responsible for genital warts—in the samples of 13 (6.73%) women in the case group and 28 controls (35.8%). No women in the control group but 16 women (8.2%) in the case group were infected with HPV 16 and 1 or more other types. In the case group, the prevalence of HPV infection was highest (63.7%) among the women aged 41 to 60 years; it was 19.6% among those aged 60 to 80 years; and it was 16.5% among those aged 21 to 40 years. The women aged 41 to 60 years were the most likely to harbor HPV DNA.
Table 1 Cervical cancer associations with behavioral, sociodemographic, sexual, and reproductive history.a Characteristic
Cases (n = 237)
HPV infection HPV16/18 175 (73.8) HPV6/11 13 (5.4) Other 7 (2.9) Education None 163 (68.7) Primary 54 (22.7) Some secondary 16 (6.7) High school graduate 4 (1.6) Annual income, Indian rupees b20 000 96 (40.5) 21 000–40 000 81 (34.1) >40 000 60 (25.3) Occupation Manual worker 191 (80.5) Housewife 46 (19.4) Age at first coitus, y ≤15 107 (45.1) 16–20 81 (34.1) 21–25 15 (6.3) >25 34 (14.3) Age at menarche, y b12 21 (8.8) 13–14 148 (62.4) ≥15 68 (28.6) No. of pregnancies 0 11 (4.6) 1–2 58 (24.4) 2–3 39 (16.4) ≥3 129 (54.4) Marital status Cohabiting 182 (76.7) Separated 48 (20.2) Widowed 7 (2.9) Frequency of sexual intercourseb Rarely 42 (17.7) ≥3 per wk 136 (57.3) 1 per wk 40 (16.8) 2 per mo 19 (8.0) No. of sexual partners 0–1 204 (86.0) ≥3 28 (13.9) No. of abortions 0 101 (42.6) 1–2 34 (14.3) ≥3 102 (43.0) Cervical screening Never 148 (62.4) 1–2 53 (22.3) ≥3 36 (15.1)
Controls (n = 257) 40 (15.5) 28 (10.8) 10 (3.8) 54 85 109 13
(21.0) (33.0) (42.4) (5.0)
OR (95% CI)
P value
15.30 (9.81–23.80) 0.40 (0.23–0.93) 0.70 (0.28–2.00)
b0.001 0.53 0.40
8.28 0.597 0.27 0.32
(5.51–12.43) (0.40–0.89) (0.15–0.49) (0.10–1.00)
0.004 0.45 0.60 0.57
137 (53.3) 62 (24.1) 58 (22.5)
0.59 (0.42–0.85) 1.63 (1.10–2.41) 1.10 (0.77–1.76)
0.44 0.20 0.29
46 (17.8) 191 (74.3)
14.9 (9.62–23.08) 0.06 (0.04–0.10)
0.001 0.81
42 126 38 51
4.21 0.33 0.67 0.38
(3.82–8.82) (0.22–0.48) (0.42–1.08) (0.20–0.72)
0.04 0.57 0.41 0.54
46 (17.8) 73 (28.4) 138 (53.6)
0.44 (0.25–0.77) 4.19 (2.87–6.11) 0.34 (0.23–0.50)
0.51 0.04 0.56
13 105 64 15
0.91 0.49 0.59 19.2
(16.3) (49.0) (14.7) (19.8)
(5.0) (40.8) (24.9) (5.8)
149 (57.9) 87 (33.8) 61 (23.7) 77 43 53 84
(29.9) (16.7) (20.6) (32.6)
(0.40–2.08) (0.31–0.69) (0.38–0.92) (10.78–34.44)
0.34 0.48 0.44 b0.001
2.39 (1.62–3.54) 0.49 (0.32–0.74) 0.09 (0.04–0.21)
0.12 0.48 0.76
0.82 6.70 1.74 0.43
0.36 0.01 0.19 0.51
(0.55–1.22) (4.41–10.16) (1.16–2.6) (0.24–0.76)
238 (92.6) 19 (7.3)
0.58 (0.32–1.09) 1.67 (0.91–3.09)
0.45 0.20
109 (42.4) 111 (43.1) 37 (14.3)
1.00 (0.70–1.44) 0.22 (0.14–0.34) 4.49 (2.91–6.92)
0.32 0.65 0.03
32 (12.4) 86 (33.4) 139 (54.0)
11.60 (7.42–18.41) 0.80 (0.60–1.29) 0.15 (0.09–0.23)
0.007 0.37 0.70
Abbreviations: CI, confidence interval; OR, odds ratio. a Values are given as number (percentage) unless otherwise indicated.
4. Discussion Invasive cervical cancer was found to be the most frequent cancer among women, and infection with HPV to be the major cause for its development [16]. Most studies have shown HPV prevalence to be Table 2 HPV infection and its association with cervical cancer. HPV Infection
Cases(n = 237) Controls(n = 257) OR (95% CI)
HPV infection, any type Positive 193 Negative 44 HPV 16 159 HPV 18 16 HPV 16-related types 21
78 179 23 17 0
10.66 (6.6–15.34) 0.01 (0.06–0.15) 11.18 (6.06–20.61) 0.32 (0.15–0.68) NA
Abbreviations: CI, confidence interval; NA, not applicable; OR, odds ratio.
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Fig. 1. PCR-based detection of HPV infection. (A) Polymerase chain reaction (PCR) amplification of the HPV L1 capsid protein gene using MY09 and MY11 primers: lane M, 1-kb DNA marker showing 7 PCR amplicons of 450 bp from cervical carcinomas. (B) Nested PCR amplification of the HPV L1 gene using the degenerate primer pair GP5+/6+: lane M, 100-bp DNA marker showing 7 PCR amplicons of 150 bp. (C) PCR amplification of HPV 16 using type-specific primers: lane M, 1-kb DNA marker showing 5 PCR amplicons of 216 bp. (D) PCR amplification of HPV 18 using type-specific primers: lane M, 100-bp DNA marker showing 7 PCR amplicons of 118 bp. (E) PCR amplification of HPV 6 and HPV 11 with consensus primers: lane M, 100-bp DNA marker showing 7 PCR amplicons of 68 bp.
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The prevalence of HPV infection increased from age group to age group until the 41- to 60-year-old group, after which it declined. A second peak concerning women older than 65 years has been reported elsewhere [6,21] but was not observed in the present study. There was therefore no reactivation of latent viruses among the study population. The same decrease in HPV prevalence in populations of increasing age has been reported in Asia [6]. Many epidemiologic studies have reported a large number of sexual partners as a major risk factor for the development of cervical cancer [22]. However, owing to the prevailing customs, a large number of partners is very rare in India, and this factor was not found to be significant. In addition to behavioral and sexual factors, infection with HR-HPV types was an important factor for the development of cervical cancer. The participants had no adequate knowledge about cervical cancer and ways of preventing it. There are no established screening programs for cervical dysplasia in Tamil Nadu, and there is certainly an urgent need to implement one in Tiruchirapalli and run it with sufficient infrastructure, staff, and other needed resources—and complement it with HPV testing as a potential alternative to cytologic screening and a preventive measure for cervical cancer. Acknowledgments The study was supported by the Indian Council of Medical Research (No. 5/13/88/06–NCD–III). Conflict of interest The authors have no conflicts of interest.
associated with several factors, which include societal economic development as well as the educational level and sexual behavior of infected persons. The prevalence of HPV infection was estimated to be 15.5% in low-income countries and 10.0% in high-income countries [17]. The present case–control study determined the distribution of HR-HPV genotypes among women with cervical cancer who live in the Tiruchirapalli district of the state of Tamil Nadu in southern India. Whereas the prevalence of HPV infection was found to be 54.9% for cases and controls combined, 82.3% of the cases, defined as women having ICC, were found to be harboring HPV DNA—a finding which was consistent with GLOBOCAN data (http://globocan.iarc. fr/factsheet.asp). Worldwide, half of all HPV infections are caused by HPV 16, 18, 31, 58, and 52. Various studies have reported HPV 16 as the most prevalent type except in eastern Africa, Japan, and Taiwan where the most common is HPV 52 [6]. In the present in study, the most common was HPV 16, for a prevalence of 29.4%. The overall prevalence of HPV 16 reported for the study population is slightly lower than the lower end of the 34.7% to 59.6% range reported in other studies [18]. In the control group, the HPV prevalence was 30.4% and the types were mostly the low-risk HPV 6 and HPV 11. The present results do not differ much from those previously reported for Tamil Nadu [19,20] and, thus, substantiate the hypothesis that HPV infection alone is not responsible for the progression of cervical cancer. A persistent infection, with integration of HPV genome into the chromosomal DNA of cervical epithelial cells, along with alterations of human oncogenes and tumor suppressor genes in the affected cells, and the impairment of immune mechanisms combine to enable the development of cervical cancer. Thus, HPV infection in women with normal cytologic findings can be attributed to the presence of genital warts, the latter sustaining the persistence of HPV infection. Infection with more than 1 genotype was detected in 8.2% of the women infected with HPV. Several studies have noted that patients could be co-infected with HPV of different genotypes. In the present study, some participants were co-infected with HPV 16 and 1 or more of other genotypes.
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