- Email: [email protected]
A population-based study of the prevalence of HPV in three districts of Tamil Nadu, India
International Journal of Gynecology and Obstetrics 129 (2015) 58–61
Contents lists available at ScienceDirect
International Journal of Gynecology and Obstetrics journal homepage: www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
A population-based study of the prevalence of HPV in three districts of Tamil Nadu, India Belli T. Sureshkumar a,b, Santhanam Shanmughapriya a, Bhudev C. Das c, Kalimuthusamy Natarajaseenivasan a,⁎ a b c
Medical Microbiology Laboratory, Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, India Department of Microbiology, Vivekananda College of Arts and Science, Tiruchengode, India Dr. BRD Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India
a r t i c l e
i n f o
Article history: Received 8 May 2014 Received in revised form 18 October 2014 Accepted 10 December 2014 Keywords: HPV HPV16 Normal cytology Prevalence Tamil Nadu
a b s t r a c t Objective: To evaluate the prevalence of HPV infection among women from three districts of Tamil Nadu (Erode, Salem, and Namakkal). Methods: Between January 27, 2007, and December 15, 2009, 1800 women aged 20–70 years who had normal cervical cytology were enrolled into a cross-sectional study. Participants completed interviews and provided first void urine samples for assessment of HPV infection. Results: Among 1699 eligible samples, 179 (10.5%) were HPV positive. HPV16 was the most common type (affecting 68 [4.0%] women), followed by HPV6 (58 [3.4%]). Among the 179 HPV-positive women, 71 (39.7%) were aged 36–45 years. An annual income of less than 25 000 INR was significantly associated with HPV infection in all three districts (P b 0.05). Regular intercourse was also associated with HPV in Erode and Salem (P b 0.05). Conclusion: Screening and prophylactic vaccines containing HPV16 and HPV6 should be promoted in western Tamil Nadu. © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cancer of the cervix is the second most common cancer among women worldwide [1]. It is the leading cancer among Indian women, but is considered a public health problem of global importance [2,3]. An estimated 493 000 new cases of cervical cancer are diagnosed worldwide every year, 80% of which are in low-income countries and nearly one-quarter in India alone [4]. The prevalence of cervical carcinoma is related to HPV prevalence in many places globally, and the presence of organized screening programs is needed to determine other factors that influence HPV infection [5]. Vaccines have been developed to combat HPV infection. The development of HPV vaccines holds tremendous promise for low-income countries like India where cervical cancer is the most common malignancy among middle-aged women, particularly in rural areas [6]. Already, there is a possibility for a HPV vaccination trial in India under the auspices of the Indian Council of Medical Research [7]. HPV16 and HPV18 have been defined as the main causes of invasive cervical cancer and its precursor lesions [8]. However, the age- and type-specific prevalence of HPV differ markedly between populations [9,10]. To maximize the cost-effectiveness of the HPV vaccination programs in India, it will
⁎ 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).
be important to understand the distribution of the major types of HPV in various regions. A previous hospital-based case–control study [11] showed an overall HPV prevalence of 54.9% in Tiruchirapalli, Tamil Nadu, India. The prevalence of HPV was 81.4% among patients with cervical carcinoma and 30.4% among women in the control group. HPV16 was found to be the most prevalent type, accounting for 82.3% of infections in the case group and 29.4% in the control group [11]. The aim of the present study was to establish the prevalence of HPV infection among women in three other districts in western Tamil Nadu and to assess the possible risk factors in the etiology of HPV infection. 2. Materials and methods The present cross-sectional study was conducted as part of a screening program to estimate the prevalence of HPV and its associated types among sexually active women with normal cytology from three districts of Tamil Nadu—Erode, Salem, and Namakkal. Between January 27, 2007, and December 15, 2009, women aged 20–70 years were identified for inclusion. Women who were unmarried, were pregnant, had undergone hysterectomy, had a history of cervical cancer, had mental illness, or had received antibiotics in the previous month were excluded. The study was approved by the Institutional Ethics Committee of Bharathidasan University (DM/07/101/373). All participants provided written informed consent. To reach women in each region, collaboration with the local healthcare centers and governmental hospitals was established. The public
http://dx.doi.org/10.1016/j.ijgo.2014.10.025 0020-7292/© 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
B.T. Sureshkumar et al. / International Journal of Gynecology and Obstetrics 129 (2015) 58–61
59
Table 1 Prevalence of HPV infection.a HPV infection
Negative Positive High risk 16 b 18 b 31 b 33 b Low risk 6b 11 b a b
Overall (n = 1699)
Erode (n = 472)
Namakkal (n = 377)
Salem (n = 850)
Single
Multiple
Total
Single
Multiple
Total
Single
Multiple
Total
Single
Multiple
Total
– 107 (6.3) 78 (5.0) 42 (2.5) 18 (1.1) 10 (0.6) 8 (0.5) 29 (1.7) 17 (1.0) 12 (0.7)
– 72 (4.2) 22 (1.3) 20 (1.2) 6 (0.4) 2 (0.1) 2 (0.1) 50 (2.9) 41 (2.4) 34 (2.0)
1520 (89.5) 179 (10.5) 100 (5.9) 68 (4.0) 24 (1.4) 12 (0.7) 10 (0.6) 79 (4.6) 58 (3.4) 46 (2.7)
– 36 (7.6) 25 (5.3) 11 (2.3) 5 (1.1) 5 (1.1) 4 (0.8) 11 (2.3) 7 (1.5) 4 (0.8)
– 55 (11.7) 10 (2.1) 8 (1.7) 2 (0.4) 2 (0.4) 2 (0.4) 45 (9.5) 33 (7.0) 30 (6.4)
381 (80.7) 91 (19.3) 35 (7.4) 19 (4.0) 7 (1.5) 7 (1.5) 6 (1.3) 56 (11.9) 40 (8.5) 34 (7.2)
– 24 (6.4) 19 (5.0) 12 (3.2) 4 (1.1) 1 (0.3) 2 (0.5) 5 (1.3) 3 (0.8) 2 (0.5)
– 5 (1.3) 4 (1.1) 3 (0.8) 1 (0.3) 0 0 1 (0.3) 2 (0.5) 2 (0.5)
348 (92.3) 29 (7.7) 23 (6.1) 15 (4.0) 5 (1.3) 1 (0.3) 2 (0.5) 6 (1.6) 5 (1.3) 4 (1.1)
– 47 (5.5) 34 (4.0) 19 (2.2) 9 (1.1) 4 (0.5) 2 (0.2) 13 (1.5) 7 (0.8) 6 (0.7)
– 12 (1.4) 8 (0.9) 9 (1.1) 3 (0.4) 0 0 4 (0.5) 6 (0.7) 2 (0.2)
791 (93.1) 59 (6.9) 42 (4.9) 28 (3.3) 12 (1.4) 4 (0.5) 2 (0.2) 17 (2.0) 13 (1.5) 8 (0.9)
Values are given as number (percentage). Women can be counted more than once because of multiple infection.
health nurses recruited participants by visiting women at home. They informed the women about the risk, primary detection, and prevention of cervical carcinoma. Participants were interviewed at the time of recruitment by trained personnel with a standard questionnaire to obtain detailed information on their sociodemographic and reproductive histories to assess the risk factors for HPV infection. Visual inspection of the cervix was done for all participants and a smear was assessed by cytoscreeners to confirm normal cytology. First void urine samples were collected from all participants in 50 mL falcon tubes and immediately transported on ice to the Microbiology laboratory of Vivekananda College of Arts and Science, Tiruchengode, India. The samples were refrigerated and stored at 4 °C before processing. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded and the resultant pellets were dissolved in 1 mL ice-cold 1X phosphate buffered saline before centrifugation at 13 000 rpm for 15 min. The resulting cell pellet was stored at − 20 °C. DNA was extracted from the pellets using the standard procedures with some modifications [12]. The quality and integrity of DNA was tested using human genomic β-globulin gene specific primers. DNA samples were tested by the MY09/11 PCR protocol [13,14]. The samples negative for MY09/11 were further assessed for the presence of HPV DNA with the general GP5+/6+ PCR system [15]. For HPV typing, type-specific (HPV16, HPV18, HPV6, and HPV11) and consensus primers (PU-1 M/PU-2R and PU31B; specific for other high-risk HPV types [HPV31, HPV33, HPV52b, and HPV58]) were used. The amplified PCR products were visualized on 1% agarose gel stained with ethidium bromide (0.5 μg/mL). Erode contains five taluks (administrative divisions), Namakkal four taluks, and Salem nine taluks. For HPV typing, 100 samples were collected from each taluk, making a total of 500 from Erode, 400 from Namakkal and 900 from Salem. The data were coded and analyzed using SPSS version 17.0 (SPSS Inc, Chicago, IL, USA). Unconditional logistic regression was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs).
The most common type of HPV detected was HPV16, followed by HPV6 (Table 1). Four different high-risk HPV types and two low-risk HPV types were detected. Multiple HPV infection was detected in 72 (4.2%) samples overall, 55 (11.7%) in Erode, 5 (1.3%) in Namakkal, and 12 (1.4%) in Salem. The most common types of multiple infections were HPV16 with HPV18 and HPV6 with HPV11 (data not shown). Among the 179 HPV-positive women, 71 (39.7%) were aged 36–45 years (Fig. 1). In Erode, frequency of HPV infection was similar among women aged 36–45 years and those aged 46–55 years. In Salem and Namakkal, the number of cases was highest among women aged 36–45 years. In Erode, HPV infection was associated with early age at marriage (≤ 18 years), annual income of less than 25 000 INR, early age at first coitus, and regular sexual intercourse (Table 2). In Namakkal, HPV infection was associated with a low income and first coitus at a very early age (b 16 years) (Table 2). Additionally, women reporting regular sexual intercourse were at increased risk, although this association was not significant (Table 2). In Salem, HPV was associated with low annual income, high parity (≥3), and regular sexual intercourse (Table 2). No associations between HPV infection and parity (with the exception of Salem) or residential area were observed (Table 2).
4. Discussion The present study has shown that 10.5% of women from three districts of Tamil Nadu who have not been previously screened for HPV have this infection. HPV16 was the most prevalent type, followed by HPV6. The prevalence of HPV infection increased with age to a peak at
3. Results Of the 1800 samples randomly selected, cell pellets were missing for 66 (18 from Erode, 15 from Namakkal, and 33 from Salem). Among the other 1734, 35 samples were discarded because they were negative for β-globulin (10 Erode, 8 Namakkal, and 17 Salem). The final 1699 samples (472 Erode, 377 Namakkal, and 850 Salem) were confirmed to have normal cervical cytology by cytology screening. Overall, 179 samples (10.5%) were HPV positive; 91 (19.3%) were positive in Erode, 29 (7.7%) in Namakkal, and 59 (6.9%) in Salem. High-risk HPV was detected in 100 (5.9%) samples overall, 35 (7.4%) in Erode, 23 (6.1%) in Namakkal, and 42 (4.9%) in Salem. Low-risk HPV was detected in 79 (4.6%) samples overall, 56 (11.9%) in Erode, 6 (1.6%) in Namakkal, and 17 (2.0%) in Salem.
Fig. 1. Number of HPV infections among women with normal cytology, by age group.
60
B.T. Sureshkumar et al. / International Journal of Gynecology and Obstetrics 129 (2015) 58–61
Table 2 Unconditional logistic regression.a Variables
Marital status Married Separated Age at marriage, y b16 16–18 19–21 N21 Residence Rural Urban Annual income, INR N25,000 b25,000 Parity 1 2 3 ≥4 Age at first coitus, y b16 16–18 19–21 N21 Intercourse d Often Rare
Erode (n = 472)
Namakkal (n = 377)
Salem (n = 820)
HPV positive (n = 91)
HPV negative (n = 381)
OR (95% CI)
HPV positive (n = 29)
HPV negative (n = 348)
OR (95% CI)
HPV positive (n = 59)
HPV negative (n = 761)
OR (95% CI)
78 (85.7) 13 (14.3)
352 (92.4) 29 (7.6)
1.00 0.49 (0.25–0.99)
27 (93.1) 2 (6.9)
289 (83.0) 59 (17.0)
1.00 0.36 (0.08–1.57)
56 (94.9) 3 (5.1)
726 (95.4) 35 (4.6)
1.00 1.11 (0.33–3.73)
7 (7.7) 42 (46.2) 10 (11.0) 32 (35.2)
10 (2.6) 95 (24.9) 145 (38.1) 131 (34.4)
3.09 (1.14–8.36)b 2.58 (1.61–4.14)b 0.21 (0.11–0.42) 1.00
3 (10.3) 12 (41.4) 2 (6.9) 12 (41.4)
29 (8.3) 136 (39.1) 23 (6.6) 160 (46.0)
1.27 (0.36–4.45) 1.10 (0.51–2.38) 1.05 (0.23–4.68) 1.00
10 (16.9) 26 (44.1) 16 (27.1) 7 (11.9)
81 (10.6) 291 (38.2) 268 (35.2) 121 (15.9)
1.71 (0.84–3.51) 1.27 (0.75-2.17) 0.68 (0.38–1.24) 1.00
42 (46.2) 49 (53.8)
177 (46.5) 204 (53.5)
0.98 (0.62–1.56) 1.00
14 (48.3) 15 (51.7)
147 (42.2) 201 (57.8)
1.28 (0.59–2.73) 1.00
46 (78.0) 13 (22.0)
519 (68.2) 242 (31.8)
1.65 (0.88–3.11) 1.00
14 (15.4) 77 (84.6)
201 (52.8) 180 (47.2)
1.00 6.14 (3.36–11.23)b
5 (17.2) 24 (82.8)
99 (28.4) 249 (71.6)
1.00 1.91 (0.71–5.14)b
23 (39.0) 36 (61.0)
435 (57.2) 326 (42.8)
1.00 2.09 (1.21–3.59)b
14 (15.4) 59 (64.8) 11 (12.1) 7 (7.7)
237 (62.2) 72 (18.9) 44 (11.5) 28 (7.3)
0.11 (0.06–0.20) 1.00 1.05 (0.44–2.49) 1.05 (0.52–2.13)
1 (3.4) 9 (31.0) 17 (58.6) 2 (6.9)
40 (11.5) 110 (31.6) 183 (52.6) 15 (4.3)
0.28 (0.04–2.08) 1.00 1.28 (0.59–2.75) 1.64 (0.36–7.57)
3 (5.1) 10 (16.9) 7 (11.9) 39 (66.1)
121 (15.9) 251 (33.0) 46 (6.0) 343 (45.1)
0.28 (0.87–0.92) 1.00 2.09 (0.91–4.86)b 2.38 (1.36–4.15)b
7 (7.7) 70 (76.9) 11 (12.1) 3 (3.3)
31 (8.1) 44 (11.5) 299 (78.5) 7 (1.8)
25.51 (14.29–45.6)c 15.25 (15.41–22.21)c 1.64 (1.02–2.07) 1.00
2 (6.9) 6 (20.7) 6 (20.7) 15 (51.7)
7 (2.0) 22 (6.3) 36 (10.3) 283 (81.3)
5.08 (0. 94–27.42)b 3.87 (1.43–10.47) 2.49 (0.95–6.55) 1.00
3 (5.1) 7 (11.9) 46 (78.0) 3 (5.1)
23 (3.0) 69 (9.1) 611 (80.3) 58 (7.6)
1.72 (0.52–5.91) 1.35 (0.59–3.09) 0.87 (0.46–1.65) 1.00
56 (61.5) 35 (38.5)
88 (23.1) 293 (76.9)
5.53 (3.28–8.65)b 1.00
23 (79.3) 6 (20.6)
247 (71.0) 101 (29.0)
1.57 (0.62–3.96) 1.00
16 (27.1) 43 (72.9)
608 (79.9) 153 (20.1)
10.68 (5.86–19.47)c 1.00
Abbreviations: OR, odds ratio; CI, confidence interval. a Values are given as number (percentage) unless indicated otherwise. b P b 0.05. c P b 0.001. d Often was defined as ≥3 times per week; rare was defined as 0–2 per week.
36–45 years, after which it fell. As expected, HPV infection was associated with economic status and sexual behavior: Early age at marriage, regular sexual intercourse, and early age at first coitus were associated with HPV positivity. Therefore, sexual behavior has a major role in HPV infection. According to the groupings of Clifford et al. [9], the overall prevalence in the three regions can be classified as intermediate, because it was similar to the overall prevalence recorded in Thailand (9.1%) [16], Vietnam (10.6%) [17], Indonesia (11.3%) [18], Chile (14.0%) [19], and Mexico (14.5%) [20]. The prevalence of high-risk HPV in the present study (5.9%) is lower than was reported in two large population-based studies of cytologically normal women in other parts of India: 10.3% of women sampled in Osmanabad District had high-risk HPV infection [21], as did 9.6% of women in Dindigul District [22]. Similarly, a previous study from Tamil Nadu [11] reported that 30.4% of women sampled had highrisk HPV infection. The fact that the prevalence was much lower in the present study could be attributable to differences in the geographical location and the social and sexual behaviors of the study populations. Various studies have shown that HPV16 is the most prevalent type worldwide, with the exception of eastern Africa, Japan, and Taiwan (where the most common type is HPV52) [8]. The overall prevalence of HPV16 in the present study was much lower than that in other studies, which have reported a prevalence of 34.7%–59.6% [23]. However, the results of the present study have shown that HPV16 and HPV6 have a predominant role in cervical cancer in western Tamil Nadu. Therefore, a prophylactic HPV vaccine containing HPV16 and HPV6 would seem appropriate to combat cervical carcinoma among the study population. In terms of the age distribution, previous studies have shown a second peak in HPV prevalence after the age of 65 years [8,15]. However, this finding was not reported in the present study, which indicates
that there is no reactivation of latent viruses among the study population. The same trend of a continued decrease in HPV prevalence with increases in age has been reported previously in Tamil Nadu [11]. An inverse association between HPV prevalence and age has also been reported in some high-income countries [10]. It is the authors’ opinion that the reproductive period of most women in India usually ends at the age of 30–35 years, which could explain why the prevalence decreased with age in the present study. In the present study, the prevalence and distribution of HPV types was assessed among sexually active women with normal cytology. To increase the overall proportion of women participating in the screening program, a non-invasive self-sampling method was adopted. Several reports have shown that self-obtained vaginal samples are an appropriate alternative for clinical cervical samples outside pregnancy [24,25]. Additionally, PCR-based HPV detection in first voided urine samples versus cervical samples showed a high sensitivity of 98.6% and a specificity of 97.4% [25]. In conclusion, HPV16 was found to be the most prevalent type of HPV in the present study, followed by HPV6. Therefore, HPV16 and HPV6 should be included in the prophylactic HPV vaccine when it is introduced to western Tamil Nadu.
Acknowledgments The Indian Council of Medical Research provided financial support (5/13/88/06/ NCD–III). Conflict of interest The authors have no conflicts of interest.
B.T. Sureshkumar et al. / International Journal of Gynecology and Obstetrics 129 (2015) 58–61
References [1] World Health Organization. WHO guidance note: Comprehensive cervical cancer prevention and control: A healthier future for girls and women. Geneva: World Health Organization; 2013. [2] Das BC, Sehgal A, Murthy NS, Gopalkrishna V, Sharma JK, Das DK, et al. Human papillomavirus and cervical cancer in Indian women. Lancet 1989;2(8674):1271. [3] 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. [4] 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. [5] Bosch FX, de Sanjosé S. Chapter 1: Human papillomavirus and cervical cancer— burden and assessment of causality. J Natl Cancer Inst Monogr 2003;31:3–13. [6] Chopra R. The Indian scene. J Clin Oncol 2001;19(18 Suppl.):106S–11S. [7] Sharma DC. India opens up to clinical trials of papillomavirus vaccines. Lancet Infect Dis 2006;6(2):76. [8] zur Hausen H. Papillomavirus infections—a major cause of human cancers. Biochim Biophys Acta 1996;1288(2):F55–78. [9] Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJ, Vaccarella S, et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005;366(9490):991–8. [10] Franceschi S, Herrero R, Clifford GM, Snijders PJ, Arslan A, Anh PT, et al. Variations in the age-specific curves of human papillomavirus prevalence in women worldwide. Int J Cancer 2006;119(11):2677–84. [11] Vinodhini K, Shanmughapriya S, Sanmugham S, Senthikuma G, Das BC, Natarajaseenivasan K. Prevalence of high-risk HPV and associated risk factors in cases of cervical carcinoma in Tamil Nadu, India. Int J Gynecol Obstet 2012;119(3):253–6. [12] Thilagavathi A, Shanmughapriya S, Vinodhini K, Das BC, Natarajaseenivasan K. Prevalence of human papillomavirus (HPV) among college going girls using self collected urine samples from Tiruchirappalli, Tamilnadu. Arch Gynecol Obstet 2012;286(6): 1483–6. [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: Oxford University Press; 1992. p. 131–52.
61
[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. [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] Sukvirach S, Smith JS, Tunsakul S, Muñoz N, Kesararat V, Opasatian O, et al. Population-based human papillomavirus prevalence in Lampang and Songkla, Thailand. J Infect Dis 2003;187(8):1246–56. [17] Pham TH, Nguyen TH, Herrero R, Vaccarella S, Smith JS, Nguyen Thuy TT, et al. Human papillomavirus infection among women in South and North Vietnam. Int J Cancer 2003;104(2):213–20. [18] Vet JN, de Boer MA, van den Akker BE, Siregar B, Lisnawati, Budiningsih S, et al. Prevalence of human papillomavirus in Indonesia: a population-based study in three regions. Br J Cancer 2008;99(1):214–8. [19] Ferreccio C, Prado RB, Luzoro AV, Ampuero SL, Snijders PJ, Meijer CJ, et al. Population-based prevalence and age distribution of human papillomavirus among women in Santiago, Chile. Cancer Epidemiol Biomarkers Prev 2004;13(12): 2271–6. [20] Lazcano-Ponce E, Herrero R, Muñoz N, Cruz A, Shah KV, Alonso P, et al. Epidemiology of HPV infection among Mexican women with normal cervical cytology. Int J Cancer 2001;91(3):412–20. [21] Sankarnarayanan R, Nene BM, Dinshaw KA, Mahe C, Jayant K, Shastri SS, et al. A cluster of randomized controlled trial of visual, cytology and human papillomavirus screening for cancer of the cervix in rural India. Int J Cancer 2005;116(4):617–23. [22] Franceschi S, Rajkumar R, Snijders PJ, Arslan A, Mahé C, Plummer M, et al. Papillomavirus infection in rural women in southern India. Br J Cancer 2005;92(3):601–6. [23] 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. [24] Snijders PJ, Verhoef VM, Arbyn M, Ogilvie G, Minozzi S, Banzi R, et al. High-risk HPV testing on self-sampled versus clinician-collected specimens: a review on the clinical accuracy and impact on population attendance in cervical cancer screening. Int J Cancer 2013;132(10):2223–36. [25] Tanzi E, Bianchi S, Fasolo MM, Frati ER, Mazza F, Martinelli M, et al. High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping. J Med Virol 2013;85(1):91–8.
Contents lists available at ScienceDirect
International Journal of Gynecology and Obstetrics journal homepage: www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
A population-based study of the prevalence of HPV in three districts of Tamil Nadu, India Belli T. Sureshkumar a,b, Santhanam Shanmughapriya a, Bhudev C. Das c, Kalimuthusamy Natarajaseenivasan a,⁎ a b c
Medical Microbiology Laboratory, Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, India Department of Microbiology, Vivekananda College of Arts and Science, Tiruchengode, India Dr. BRD Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India
a r t i c l e
i n f o
Article history: Received 8 May 2014 Received in revised form 18 October 2014 Accepted 10 December 2014 Keywords: HPV HPV16 Normal cytology Prevalence Tamil Nadu
a b s t r a c t Objective: To evaluate the prevalence of HPV infection among women from three districts of Tamil Nadu (Erode, Salem, and Namakkal). Methods: Between January 27, 2007, and December 15, 2009, 1800 women aged 20–70 years who had normal cervical cytology were enrolled into a cross-sectional study. Participants completed interviews and provided first void urine samples for assessment of HPV infection. Results: Among 1699 eligible samples, 179 (10.5%) were HPV positive. HPV16 was the most common type (affecting 68 [4.0%] women), followed by HPV6 (58 [3.4%]). Among the 179 HPV-positive women, 71 (39.7%) were aged 36–45 years. An annual income of less than 25 000 INR was significantly associated with HPV infection in all three districts (P b 0.05). Regular intercourse was also associated with HPV in Erode and Salem (P b 0.05). Conclusion: Screening and prophylactic vaccines containing HPV16 and HPV6 should be promoted in western Tamil Nadu. © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cancer of the cervix is the second most common cancer among women worldwide [1]. It is the leading cancer among Indian women, but is considered a public health problem of global importance [2,3]. An estimated 493 000 new cases of cervical cancer are diagnosed worldwide every year, 80% of which are in low-income countries and nearly one-quarter in India alone [4]. The prevalence of cervical carcinoma is related to HPV prevalence in many places globally, and the presence of organized screening programs is needed to determine other factors that influence HPV infection [5]. Vaccines have been developed to combat HPV infection. The development of HPV vaccines holds tremendous promise for low-income countries like India where cervical cancer is the most common malignancy among middle-aged women, particularly in rural areas [6]. Already, there is a possibility for a HPV vaccination trial in India under the auspices of the Indian Council of Medical Research [7]. HPV16 and HPV18 have been defined as the main causes of invasive cervical cancer and its precursor lesions [8]. However, the age- and type-specific prevalence of HPV differ markedly between populations [9,10]. To maximize the cost-effectiveness of the HPV vaccination programs in India, it will
⁎ 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).
be important to understand the distribution of the major types of HPV in various regions. A previous hospital-based case–control study [11] showed an overall HPV prevalence of 54.9% in Tiruchirapalli, Tamil Nadu, India. The prevalence of HPV was 81.4% among patients with cervical carcinoma and 30.4% among women in the control group. HPV16 was found to be the most prevalent type, accounting for 82.3% of infections in the case group and 29.4% in the control group [11]. The aim of the present study was to establish the prevalence of HPV infection among women in three other districts in western Tamil Nadu and to assess the possible risk factors in the etiology of HPV infection. 2. Materials and methods The present cross-sectional study was conducted as part of a screening program to estimate the prevalence of HPV and its associated types among sexually active women with normal cytology from three districts of Tamil Nadu—Erode, Salem, and Namakkal. Between January 27, 2007, and December 15, 2009, women aged 20–70 years were identified for inclusion. Women who were unmarried, were pregnant, had undergone hysterectomy, had a history of cervical cancer, had mental illness, or had received antibiotics in the previous month were excluded. The study was approved by the Institutional Ethics Committee of Bharathidasan University (DM/07/101/373). All participants provided written informed consent. To reach women in each region, collaboration with the local healthcare centers and governmental hospitals was established. The public
http://dx.doi.org/10.1016/j.ijgo.2014.10.025 0020-7292/© 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
B.T. Sureshkumar et al. / International Journal of Gynecology and Obstetrics 129 (2015) 58–61
59
Table 1 Prevalence of HPV infection.a HPV infection
Negative Positive High risk 16 b 18 b 31 b 33 b Low risk 6b 11 b a b
Overall (n = 1699)
Erode (n = 472)
Namakkal (n = 377)
Salem (n = 850)
Single
Multiple
Total
Single
Multiple
Total
Single
Multiple
Total
Single
Multiple
Total
– 107 (6.3) 78 (5.0) 42 (2.5) 18 (1.1) 10 (0.6) 8 (0.5) 29 (1.7) 17 (1.0) 12 (0.7)
– 72 (4.2) 22 (1.3) 20 (1.2) 6 (0.4) 2 (0.1) 2 (0.1) 50 (2.9) 41 (2.4) 34 (2.0)
1520 (89.5) 179 (10.5) 100 (5.9) 68 (4.0) 24 (1.4) 12 (0.7) 10 (0.6) 79 (4.6) 58 (3.4) 46 (2.7)
– 36 (7.6) 25 (5.3) 11 (2.3) 5 (1.1) 5 (1.1) 4 (0.8) 11 (2.3) 7 (1.5) 4 (0.8)
– 55 (11.7) 10 (2.1) 8 (1.7) 2 (0.4) 2 (0.4) 2 (0.4) 45 (9.5) 33 (7.0) 30 (6.4)
381 (80.7) 91 (19.3) 35 (7.4) 19 (4.0) 7 (1.5) 7 (1.5) 6 (1.3) 56 (11.9) 40 (8.5) 34 (7.2)
– 24 (6.4) 19 (5.0) 12 (3.2) 4 (1.1) 1 (0.3) 2 (0.5) 5 (1.3) 3 (0.8) 2 (0.5)
– 5 (1.3) 4 (1.1) 3 (0.8) 1 (0.3) 0 0 1 (0.3) 2 (0.5) 2 (0.5)
348 (92.3) 29 (7.7) 23 (6.1) 15 (4.0) 5 (1.3) 1 (0.3) 2 (0.5) 6 (1.6) 5 (1.3) 4 (1.1)
– 47 (5.5) 34 (4.0) 19 (2.2) 9 (1.1) 4 (0.5) 2 (0.2) 13 (1.5) 7 (0.8) 6 (0.7)
– 12 (1.4) 8 (0.9) 9 (1.1) 3 (0.4) 0 0 4 (0.5) 6 (0.7) 2 (0.2)
791 (93.1) 59 (6.9) 42 (4.9) 28 (3.3) 12 (1.4) 4 (0.5) 2 (0.2) 17 (2.0) 13 (1.5) 8 (0.9)
Values are given as number (percentage). Women can be counted more than once because of multiple infection.
health nurses recruited participants by visiting women at home. They informed the women about the risk, primary detection, and prevention of cervical carcinoma. Participants were interviewed at the time of recruitment by trained personnel with a standard questionnaire to obtain detailed information on their sociodemographic and reproductive histories to assess the risk factors for HPV infection. Visual inspection of the cervix was done for all participants and a smear was assessed by cytoscreeners to confirm normal cytology. First void urine samples were collected from all participants in 50 mL falcon tubes and immediately transported on ice to the Microbiology laboratory of Vivekananda College of Arts and Science, Tiruchengode, India. The samples were refrigerated and stored at 4 °C before processing. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded and the resultant pellets were dissolved in 1 mL ice-cold 1X phosphate buffered saline before centrifugation at 13 000 rpm for 15 min. The resulting cell pellet was stored at − 20 °C. DNA was extracted from the pellets using the standard procedures with some modifications [12]. The quality and integrity of DNA was tested using human genomic β-globulin gene specific primers. DNA samples were tested by the MY09/11 PCR protocol [13,14]. The samples negative for MY09/11 were further assessed for the presence of HPV DNA with the general GP5+/6+ PCR system [15]. For HPV typing, type-specific (HPV16, HPV18, HPV6, and HPV11) and consensus primers (PU-1 M/PU-2R and PU31B; specific for other high-risk HPV types [HPV31, HPV33, HPV52b, and HPV58]) were used. The amplified PCR products were visualized on 1% agarose gel stained with ethidium bromide (0.5 μg/mL). Erode contains five taluks (administrative divisions), Namakkal four taluks, and Salem nine taluks. For HPV typing, 100 samples were collected from each taluk, making a total of 500 from Erode, 400 from Namakkal and 900 from Salem. The data were coded and analyzed using SPSS version 17.0 (SPSS Inc, Chicago, IL, USA). Unconditional logistic regression was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs).
The most common type of HPV detected was HPV16, followed by HPV6 (Table 1). Four different high-risk HPV types and two low-risk HPV types were detected. Multiple HPV infection was detected in 72 (4.2%) samples overall, 55 (11.7%) in Erode, 5 (1.3%) in Namakkal, and 12 (1.4%) in Salem. The most common types of multiple infections were HPV16 with HPV18 and HPV6 with HPV11 (data not shown). Among the 179 HPV-positive women, 71 (39.7%) were aged 36–45 years (Fig. 1). In Erode, frequency of HPV infection was similar among women aged 36–45 years and those aged 46–55 years. In Salem and Namakkal, the number of cases was highest among women aged 36–45 years. In Erode, HPV infection was associated with early age at marriage (≤ 18 years), annual income of less than 25 000 INR, early age at first coitus, and regular sexual intercourse (Table 2). In Namakkal, HPV infection was associated with a low income and first coitus at a very early age (b 16 years) (Table 2). Additionally, women reporting regular sexual intercourse were at increased risk, although this association was not significant (Table 2). In Salem, HPV was associated with low annual income, high parity (≥3), and regular sexual intercourse (Table 2). No associations between HPV infection and parity (with the exception of Salem) or residential area were observed (Table 2).
4. Discussion The present study has shown that 10.5% of women from three districts of Tamil Nadu who have not been previously screened for HPV have this infection. HPV16 was the most prevalent type, followed by HPV6. The prevalence of HPV infection increased with age to a peak at
3. Results Of the 1800 samples randomly selected, cell pellets were missing for 66 (18 from Erode, 15 from Namakkal, and 33 from Salem). Among the other 1734, 35 samples were discarded because they were negative for β-globulin (10 Erode, 8 Namakkal, and 17 Salem). The final 1699 samples (472 Erode, 377 Namakkal, and 850 Salem) were confirmed to have normal cervical cytology by cytology screening. Overall, 179 samples (10.5%) were HPV positive; 91 (19.3%) were positive in Erode, 29 (7.7%) in Namakkal, and 59 (6.9%) in Salem. High-risk HPV was detected in 100 (5.9%) samples overall, 35 (7.4%) in Erode, 23 (6.1%) in Namakkal, and 42 (4.9%) in Salem. Low-risk HPV was detected in 79 (4.6%) samples overall, 56 (11.9%) in Erode, 6 (1.6%) in Namakkal, and 17 (2.0%) in Salem.
Fig. 1. Number of HPV infections among women with normal cytology, by age group.
60
B.T. Sureshkumar et al. / International Journal of Gynecology and Obstetrics 129 (2015) 58–61
Table 2 Unconditional logistic regression.a Variables
Marital status Married Separated Age at marriage, y b16 16–18 19–21 N21 Residence Rural Urban Annual income, INR N25,000 b25,000 Parity 1 2 3 ≥4 Age at first coitus, y b16 16–18 19–21 N21 Intercourse d Often Rare
Erode (n = 472)
Namakkal (n = 377)
Salem (n = 820)
HPV positive (n = 91)
HPV negative (n = 381)
OR (95% CI)
HPV positive (n = 29)
HPV negative (n = 348)
OR (95% CI)
HPV positive (n = 59)
HPV negative (n = 761)
OR (95% CI)
78 (85.7) 13 (14.3)
352 (92.4) 29 (7.6)
1.00 0.49 (0.25–0.99)
27 (93.1) 2 (6.9)
289 (83.0) 59 (17.0)
1.00 0.36 (0.08–1.57)
56 (94.9) 3 (5.1)
726 (95.4) 35 (4.6)
1.00 1.11 (0.33–3.73)
7 (7.7) 42 (46.2) 10 (11.0) 32 (35.2)
10 (2.6) 95 (24.9) 145 (38.1) 131 (34.4)
3.09 (1.14–8.36)b 2.58 (1.61–4.14)b 0.21 (0.11–0.42) 1.00
3 (10.3) 12 (41.4) 2 (6.9) 12 (41.4)
29 (8.3) 136 (39.1) 23 (6.6) 160 (46.0)
1.27 (0.36–4.45) 1.10 (0.51–2.38) 1.05 (0.23–4.68) 1.00
10 (16.9) 26 (44.1) 16 (27.1) 7 (11.9)
81 (10.6) 291 (38.2) 268 (35.2) 121 (15.9)
1.71 (0.84–3.51) 1.27 (0.75-2.17) 0.68 (0.38–1.24) 1.00
42 (46.2) 49 (53.8)
177 (46.5) 204 (53.5)
0.98 (0.62–1.56) 1.00
14 (48.3) 15 (51.7)
147 (42.2) 201 (57.8)
1.28 (0.59–2.73) 1.00
46 (78.0) 13 (22.0)
519 (68.2) 242 (31.8)
1.65 (0.88–3.11) 1.00
14 (15.4) 77 (84.6)
201 (52.8) 180 (47.2)
1.00 6.14 (3.36–11.23)b
5 (17.2) 24 (82.8)
99 (28.4) 249 (71.6)
1.00 1.91 (0.71–5.14)b
23 (39.0) 36 (61.0)
435 (57.2) 326 (42.8)
1.00 2.09 (1.21–3.59)b
14 (15.4) 59 (64.8) 11 (12.1) 7 (7.7)
237 (62.2) 72 (18.9) 44 (11.5) 28 (7.3)
0.11 (0.06–0.20) 1.00 1.05 (0.44–2.49) 1.05 (0.52–2.13)
1 (3.4) 9 (31.0) 17 (58.6) 2 (6.9)
40 (11.5) 110 (31.6) 183 (52.6) 15 (4.3)
0.28 (0.04–2.08) 1.00 1.28 (0.59–2.75) 1.64 (0.36–7.57)
3 (5.1) 10 (16.9) 7 (11.9) 39 (66.1)
121 (15.9) 251 (33.0) 46 (6.0) 343 (45.1)
0.28 (0.87–0.92) 1.00 2.09 (0.91–4.86)b 2.38 (1.36–4.15)b
7 (7.7) 70 (76.9) 11 (12.1) 3 (3.3)
31 (8.1) 44 (11.5) 299 (78.5) 7 (1.8)
25.51 (14.29–45.6)c 15.25 (15.41–22.21)c 1.64 (1.02–2.07) 1.00
2 (6.9) 6 (20.7) 6 (20.7) 15 (51.7)
7 (2.0) 22 (6.3) 36 (10.3) 283 (81.3)
5.08 (0. 94–27.42)b 3.87 (1.43–10.47) 2.49 (0.95–6.55) 1.00
3 (5.1) 7 (11.9) 46 (78.0) 3 (5.1)
23 (3.0) 69 (9.1) 611 (80.3) 58 (7.6)
1.72 (0.52–5.91) 1.35 (0.59–3.09) 0.87 (0.46–1.65) 1.00
56 (61.5) 35 (38.5)
88 (23.1) 293 (76.9)
5.53 (3.28–8.65)b 1.00
23 (79.3) 6 (20.6)
247 (71.0) 101 (29.0)
1.57 (0.62–3.96) 1.00
16 (27.1) 43 (72.9)
608 (79.9) 153 (20.1)
10.68 (5.86–19.47)c 1.00
Abbreviations: OR, odds ratio; CI, confidence interval. a Values are given as number (percentage) unless indicated otherwise. b P b 0.05. c P b 0.001. d Often was defined as ≥3 times per week; rare was defined as 0–2 per week.
36–45 years, after which it fell. As expected, HPV infection was associated with economic status and sexual behavior: Early age at marriage, regular sexual intercourse, and early age at first coitus were associated with HPV positivity. Therefore, sexual behavior has a major role in HPV infection. According to the groupings of Clifford et al. [9], the overall prevalence in the three regions can be classified as intermediate, because it was similar to the overall prevalence recorded in Thailand (9.1%) [16], Vietnam (10.6%) [17], Indonesia (11.3%) [18], Chile (14.0%) [19], and Mexico (14.5%) [20]. The prevalence of high-risk HPV in the present study (5.9%) is lower than was reported in two large population-based studies of cytologically normal women in other parts of India: 10.3% of women sampled in Osmanabad District had high-risk HPV infection [21], as did 9.6% of women in Dindigul District [22]. Similarly, a previous study from Tamil Nadu [11] reported that 30.4% of women sampled had highrisk HPV infection. The fact that the prevalence was much lower in the present study could be attributable to differences in the geographical location and the social and sexual behaviors of the study populations. Various studies have shown that HPV16 is the most prevalent type worldwide, with the exception of eastern Africa, Japan, and Taiwan (where the most common type is HPV52) [8]. The overall prevalence of HPV16 in the present study was much lower than that in other studies, which have reported a prevalence of 34.7%–59.6% [23]. However, the results of the present study have shown that HPV16 and HPV6 have a predominant role in cervical cancer in western Tamil Nadu. Therefore, a prophylactic HPV vaccine containing HPV16 and HPV6 would seem appropriate to combat cervical carcinoma among the study population. In terms of the age distribution, previous studies have shown a second peak in HPV prevalence after the age of 65 years [8,15]. However, this finding was not reported in the present study, which indicates
that there is no reactivation of latent viruses among the study population. The same trend of a continued decrease in HPV prevalence with increases in age has been reported previously in Tamil Nadu [11]. An inverse association between HPV prevalence and age has also been reported in some high-income countries [10]. It is the authors’ opinion that the reproductive period of most women in India usually ends at the age of 30–35 years, which could explain why the prevalence decreased with age in the present study. In the present study, the prevalence and distribution of HPV types was assessed among sexually active women with normal cytology. To increase the overall proportion of women participating in the screening program, a non-invasive self-sampling method was adopted. Several reports have shown that self-obtained vaginal samples are an appropriate alternative for clinical cervical samples outside pregnancy [24,25]. Additionally, PCR-based HPV detection in first voided urine samples versus cervical samples showed a high sensitivity of 98.6% and a specificity of 97.4% [25]. In conclusion, HPV16 was found to be the most prevalent type of HPV in the present study, followed by HPV6. Therefore, HPV16 and HPV6 should be included in the prophylactic HPV vaccine when it is introduced to western Tamil Nadu.
Acknowledgments The Indian Council of Medical Research provided financial support (5/13/88/06/ NCD–III). Conflict of interest The authors have no conflicts of interest.
B.T. Sureshkumar et al. / International Journal of Gynecology and Obstetrics 129 (2015) 58–61
References [1] World Health Organization. WHO guidance note: Comprehensive cervical cancer prevention and control: A healthier future for girls and women. Geneva: World Health Organization; 2013. [2] Das BC, Sehgal A, Murthy NS, Gopalkrishna V, Sharma JK, Das DK, et al. Human papillomavirus and cervical cancer in Indian women. Lancet 1989;2(8674):1271. [3] 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. [4] 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. [5] Bosch FX, de Sanjosé S. Chapter 1: Human papillomavirus and cervical cancer— burden and assessment of causality. J Natl Cancer Inst Monogr 2003;31:3–13. [6] Chopra R. The Indian scene. J Clin Oncol 2001;19(18 Suppl.):106S–11S. [7] Sharma DC. India opens up to clinical trials of papillomavirus vaccines. Lancet Infect Dis 2006;6(2):76. [8] zur Hausen H. Papillomavirus infections—a major cause of human cancers. Biochim Biophys Acta 1996;1288(2):F55–78. [9] Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJ, Vaccarella S, et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005;366(9490):991–8. [10] Franceschi S, Herrero R, Clifford GM, Snijders PJ, Arslan A, Anh PT, et al. Variations in the age-specific curves of human papillomavirus prevalence in women worldwide. Int J Cancer 2006;119(11):2677–84. [11] Vinodhini K, Shanmughapriya S, Sanmugham S, Senthikuma G, Das BC, Natarajaseenivasan K. Prevalence of high-risk HPV and associated risk factors in cases of cervical carcinoma in Tamil Nadu, India. Int J Gynecol Obstet 2012;119(3):253–6. [12] Thilagavathi A, Shanmughapriya S, Vinodhini K, Das BC, Natarajaseenivasan K. Prevalence of human papillomavirus (HPV) among college going girls using self collected urine samples from Tiruchirappalli, Tamilnadu. Arch Gynecol Obstet 2012;286(6): 1483–6. [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: Oxford University Press; 1992. p. 131–52.
61
[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. [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] Sukvirach S, Smith JS, Tunsakul S, Muñoz N, Kesararat V, Opasatian O, et al. Population-based human papillomavirus prevalence in Lampang and Songkla, Thailand. J Infect Dis 2003;187(8):1246–56. [17] Pham TH, Nguyen TH, Herrero R, Vaccarella S, Smith JS, Nguyen Thuy TT, et al. Human papillomavirus infection among women in South and North Vietnam. Int J Cancer 2003;104(2):213–20. [18] Vet JN, de Boer MA, van den Akker BE, Siregar B, Lisnawati, Budiningsih S, et al. Prevalence of human papillomavirus in Indonesia: a population-based study in three regions. Br J Cancer 2008;99(1):214–8. [19] Ferreccio C, Prado RB, Luzoro AV, Ampuero SL, Snijders PJ, Meijer CJ, et al. Population-based prevalence and age distribution of human papillomavirus among women in Santiago, Chile. Cancer Epidemiol Biomarkers Prev 2004;13(12): 2271–6. [20] Lazcano-Ponce E, Herrero R, Muñoz N, Cruz A, Shah KV, Alonso P, et al. Epidemiology of HPV infection among Mexican women with normal cervical cytology. Int J Cancer 2001;91(3):412–20. [21] Sankarnarayanan R, Nene BM, Dinshaw KA, Mahe C, Jayant K, Shastri SS, et al. A cluster of randomized controlled trial of visual, cytology and human papillomavirus screening for cancer of the cervix in rural India. Int J Cancer 2005;116(4):617–23. [22] Franceschi S, Rajkumar R, Snijders PJ, Arslan A, Mahé C, Plummer M, et al. Papillomavirus infection in rural women in southern India. Br J Cancer 2005;92(3):601–6. [23] 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. [24] Snijders PJ, Verhoef VM, Arbyn M, Ogilvie G, Minozzi S, Banzi R, et al. High-risk HPV testing on self-sampled versus clinician-collected specimens: a review on the clinical accuracy and impact on population attendance in cervical cancer screening. Int J Cancer 2013;132(10):2223–36. [25] Tanzi E, Bianchi S, Fasolo MM, Frati ER, Mazza F, Martinelli M, et al. High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping. J Med Virol 2013;85(1):91–8.