- Email: [email protected]
penile smear or biopsy specimens
Journal of Clinical Virology 37 (2006) 190–194
Human papillomavirus DNA in urine samples of women with or without cervical cancer and their male partners compared with simultaneously collected cervical/penile smear or biopsy specimens Amita Gupta a , Raksha Arora a , Sanjay Gupta b , Bhupesh K. Prusty c , Uma Kailash c , Swaraj Batra a , Bhudev C. Das c,∗ a Department of Gynecology and Obstetrics, Maulana Azad Medical College, Lok Nayak Hospital, New Delhi Division of Cytopathology, Institute of Cytology and Preventive Oncology (ICMR), I-7, Sector-39, Noida 201301, UP, India Division of Molecular Oncology, Institute of Cytology and Preventive Oncology (ICMR), I-7, Sector-39, Noida 201301, UP, India b
c
Received 16 December 2005; received in revised form 20 April 2006; accepted 19 July 2006
Abstract Infection of specific types of high-risk human papillomaviruses (HPVs) causes cervical cancer in women. Conventional test for genital HPV infection requires collection of scraped cervical cells or biopsy specimens, which involves invasive procedures. Utility of non-invasive urine sampling for detection of HPV in women and their male sexual partners is controversial. The validation of this urine-based HPV DNA test is of immense value not only in screening large population and children but also for HPV vaccine monitoring in adolescents. We examined the frequency of high risk HPV types 16 and 18 in simultaneously collected urine samples and cervical scrapes or biopsy specimens from women with cervical cancer and their single lifetime male sexual partners in order to validate the utility of urine sampling as a reliable non-invasive method for detection of genital HPV infection. Thirty women with invasive cervical cancer and their husbands along with 30 age-matched normal healthy women including their husbands were recruited for the study. Cervical biopsies/scrapes from women subjects and penile scrapes from their husbands and urine samples from all of them were collected before taking biopsy or scrapes. HPV-L1 consensus primer as well as high-risk HPV (HPV 16 and 18) type-specific oligo-primers were used for PCR detection of HPV DNA. The total frequency of HPV in women with cervical cancer was found to be 83% (25/30) while it was only 67% (20/30) in their male partners but there was virtually no difference in results between urine and scrape or tissue biopsy either in women or their male partners. Although healthy women and their husbands showed similar frequency of HPV infection both in urine and scrape samples, there was a significant difference (p = 0.05) in the prevalence of high risk HPV type 16 in women with cervical cancer (70%) and their male partners (30%). Similar was the trend between control women and their male partners. The results also showed a very high prevalence of HPV type 16 among Indian women with cervical cancer while its frequency was significantly low in their single lifetime male partners. The case by case matching of HPV positivity and negativity between urine and cervical/penile scrapes or biopsies obtained from women and their male partners demonstrated that the non-invasive urine sampling can be reliably used for screening genital HPV infection in both men and women. © 2006 Published by Elsevier B.V. Keywords: Human papillomavirus; Cervical cancer; Women male partner; Urine; Cervical scrapes; PCR
1. Introduction Cervical cancer is the most common cancer in Indian women with an annual incidence of about 120,000 new cases, constituting almost 16% of world’s annual incidence ∗
Corresponding author. Tel.: +91 120 2575838; fax: +91 120 2579473. E-mail address: [email protected] (B.C. Das).
1386-6532/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jcv.2006.07.007
(WHO, 1986). Several epidemiological risk factors are associated with the development of this cancer including multiple sexual partners, sexual intercourse at an early age, number of pregnancies, poor genital hygiene, smoking, oral contraceptives etc. Specific types of high risk-human papillomaviruses (HR-HPVs) have been strongly implicated as principal causal agents in the development of cervical cancer (Das et al., 1992a, 2000; Vizcano et al., 2000; Zur
A. Gupta et al. / Journal of Clinical Virology 37 (2006) 190–194
Hausen, 1979; Munoz et al., 2003). Of more than 100 HPV types thus far described, about 30 types are associated with anogenital cancers and HPV types 16 and 18 are considered to be the major high-risk HPV (HR-HPV) types. In India, as high as 98% cervical cancer cases show presence of HPV as compared to 5–20% in normal healthy controls (Das et al., 2000). HPV 16 is the most prevalent type (90%) while a very low frequency (3%) of HPV type 18 is found in India (Das et al., 1992a, 2000). Recent reports also indicate a low prevalence of other high-risk HPV types in India (Franceschi et al., 2003, 2005; Sowjanya et al., 2005). As with all other sexually transmitted diseases, the approach to control and treat HPV-associated diseases should include assessment of both the index case as well as his or her sexual partner(s). Therefore, understanding manifestations, diagnosis, and treatment of HPV-related diseases of male genital tract is an important part of caring women with genital HPV infection. Thus, screening of male sexual partners for HPV infection has been suggested for women presenting with cervical squamous intraepithelial lesions (Palefsky and Barrasso, 1996). Despite strong evidence implicating specific HR-HPV types in cervical carcinogenesis and an apparent role of male partners in causation of this disease, genital HPV infection in men has not been thoroughly investigated. In particular, the male reservoir for HPV types detected in cervical neoplasia is poorly understood. Studies using PCRbased HPV detection among male partners of women with cervical neoplasia are few and none from India where cervical cancer and infection of HPV are highest in the world and it forms a major public health problem. The present study has therefore been designed to investigate the prevalence of high risk HPV type 16 and HPV type 18 in women with cervical cancer and their male sexual partners in comparison to normal healthy women and their husbands. Conventional test for genital HPV infection requires collection of scraped cervical cells or biopsy specimens, which involves invasive procedures in a clinic. Such invasive methods are not feasible for a large-scale population screening. Thus there is a need for a simple, reliable and a non-invasive method for screening general population. Urine sampling has been utilized for detection of various genital infections (Hillman et al., 1993a; Tamin et al., 2002) including HPV infection (Brinkman et al., 2002, 2004; Das et al., 1992b; Gopalkrishna et al., 1992; Hillman et al., 1993a,b; Stanczuk et al., 2003). However, utility of urine sampling for detection of HPV infection is controversial (Powell et al., 2003; Fife et al., 2003; Lazcano-Ponce et al., 2001a,b). We have therefore compared the frequency of HPV infection between cervical and penile scrapes or tumor biopsies and urine samples of both partners to validate the utility of urine sampling as a reliable non-invasive method for detection of HPV infection. 2. Materials and methods The study group comprised 30 women with histologically confirmed invasive cervical cancer and their husbands and the
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control group consisted of 30 age-matched (±5 years) women with normal or inflammatory or negative cervical cytology and their husbands. The women having multiple male sexual partners were excluded from the study. Informed consent was taken from all enrolled subjects and Institutional Ethical Committee approved using part of human materials obtained for routine diagnostic purposes for research investigation. All male partners were interviewed regarding their sexual behavior by male interviewers using a structured questionnaire. Cervical biopsies from women with cervical cancer and scraped cervical cells from normal healthy women were collected. Cervical cells were collected by scraping the ectocervix or the surface of cervical portio with a wooden Ayre’s spatula. The spatula along with cervical materials were transferred to collection bottles containing phosphate buffered saline (PBS) and stored at −70 ◦ C deep freezer till further processing. The penile cell samples were collected by swabbing the intrameatal and distal urethra, the external surface of the glans and the coronal sulcus of the penis with wet thin cotton-tipped swabs. A smear on glass slide was also prepared for Pap test and remaining cells were eluted in PBS and stored at −70 ◦ C. Urine samples were collected from both women and their male partners before taking biopsy or cervical scrape or penile swab. DNA extraction was done by using both organic and non-organic methods (Gopalkrishna et al., 1992). The PCR amplification of HPV-DNA sequences was carried out first by L1 consensus primers and then by HPV types 16 and 18specific primers using the method of Saiki et al. with some modifications (Saiki et al., 1988). The primers used were: (HPV16 upstream 5 -AAG GCC AAC TAA ATG TCA C-3 , HPV16 downstream 5 -CTG CTT TTA TAC TAA CCG G-3 ; HPV18 upstream 5 -ACC TTA ATG AAA AAC CAC GA3 , HPV18 downstream 5 -CGT CGT TTA GAG TCG TTC CTG-3 ). All the samples were initially screened by the L1 consensus primers (L1 upstream 5 -GCM CAG GGW CAT AAY AAT GG-3 , L1 downstream 5 -CGT CCM AAR GGA WAC TGA TC-3 , where, M = A or C, R = A or G, W = A or T, Y = C or T) with beta globin gene (5 -GAA GAG CCA AGG ACA GGT AC-3 and 5 -CAA CTT CAT CCA CGT TAC ACC-3 ) as an internal control. Statistical significance was calculated using χ2 as well as κ statistics where k = 1, p < 0.001.
3. Results Comparison of various epidemiological characteristics of cervical cancer cases and their male partners along with controls is presented in Table 1. The mean age of women with cancer was 41.7 years, while 42.1 years was for the control group. The mean age of husbands of women in the study group was 46.4 years, and that of the control group was 46.9 years (see Table 1). Substantial difference was also observed in sexual behavior of male partners of cancer cases when compared to that of controls. They reported significantly a higher
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Table 1 Comparison of various epidemiological characteristics in cervical cancer cases, controls and their partners
Age mean (S.D.) Age at marriage (S.D.) Age at first coitus (S.D.) Illiterate Smoke Husband illiterate Husband smoking Husband PMSC Husband extra-MSC Circumcised
Case (n = 30) Control (n = 30) OR
P value
41.7 (6.9) 16.8 (2.3) 17.2 (2.0) 70% 50% 46.7% 86.7% 26.7% 53.3% 6.7%
0.73 <0.001 <0.001 0.004 <0.001 0.028 <0.001 0.54 <0.01 0.39
42.1 (5.9) 21.0 (2.2) 21.1 (2.2) 33.3% 10% 20% 43.3% 20% 20% 13.3%
– – – 4.7 9.0 3.5 8.5 1.4 4.6 0.46
PMSC: pre-marital sexual contacts; extra MSC: extra marital sexual contacts; OR: odds ratio.
weekly frequency of sexual intercourse, extra-marital sexual contact, contact with prostitutes and are nonuser of condoms. A majority of women with cervical cancer and their husbands were smokers, illiterate and had recorded poor genital hygiene when compared to those of healthy control women and their male partners. The frequency of circumcision in partners of cancer patients was also very low in comparison to their normal counterpart (Table 1). Out of 30 women with cervical cancer, 9 had well differentiated, 16 moderately differentiated and 4 poorly differentiated squamous cell carcinoma (SCC), and one adenocarcinoma. Of 30 controls, 15 women had cytologically negative Pap smear and 15 had negative Pap smear with inflammation. All the penile scrapes (30 each) from male partners of cases as well as controls were found to be benign anucleate or nucleate squamous cells and no cytological evidence of HPV infection could be discerned. The frequency of total HPV infection as revealed by L1 consensus primer in women with invasive cervical cancer was found to be 83.3% while it was 82.1% in the urine samples from them. DNA could not be isolated in two urine samples. Barring these two cases, there is 100% concordance for the HPV positivity and negativity between urine and cervical scrape/biopsy (Table 2). HPV 16 was the most predominant type with a prevalence of 70% in cervical biopsy specimens and 68% in urine samples by PCR, as compared to only 16.7% in both types of controls. In control group, HPV DNA was detected in the same 8 (26.7%) women both in their cervical scrapes and urine samples, demonstrating 100% agreement between them (Table 2). Four of these eight women had normal or nega-
tive Pap smear while other four had negative Pap smear with inflammation. HPV type 16 was detected in five of them in both cervical scrapes and in urine, giving prevalence of 16.7% in each. HPV 18 was not detected in any of the cervical smear or urine samples of the control group. In case of male partners of women with cervical cancer, HPV DNA detection rate was 66.7% as compared to 26.7% (Table 3) in control males and the difference was found to be statistically significant (p < 0.001). Although the prevalence of HPV was lower in male partners, 100% matching was found in HPV positivity between scraped penile cells and urine samples and HPV 16 was also the predominant type detected among the male partners of women. Concordance between the two types biological specimens i.e. cervical/penile scrapes and urine samples was estimated considering only those samples for which the results were available for both the specimens. It was found that out of 28 samples (two samples being inadequate in urine), 23 were positive for HPV infections and the rest were negative. The results of urine samples revealed positivity for all these 23 cases that also showed positivity in their scrape material. Thus the total concordance of the positivity and negativity was found to be 100% for the total HPV infections. Similar concordance was found for the high-risk HPV type 16 infections. Out of 25 HPV positive women with cervical cancer, 19 of their male partners were HPV positive resulting in non-specific concordance rate for HPV infection as 76%. In control group, out of eight HPV positive women, four of their male partners were HPV positive, giving a non-specific concordance rate of 50%. Type-specific concordance for HPV type 16 was found to be 43% in study group as nine male partners of 21 HPV-16 positive women were found to be HPV 16 positive. In control group only one male partner out of five HPV 16 positive women was found to be positive for HPV 16, resulting in type specific concordance rate of 20% for HPV 16. The difference, however, was not found to be statistically significant.
4. Discussion In the present study, the results show virtually no difference in the frequency of HPV infection between cervical scrapes/biopsies and urine samples either in women or in their male partners leading to 100% agreement for the HPV
Table 2 Percentage of HPV positivity in urine samples and its agreement with scrape or tissue samples of cervical cancers and controls along with their spouse Type of subjects
% of HPV positivity
*%
of agreement with scrape/tissues
% of total HPV positivity
* % of agreement with scrape/tissues
Cases (n = 28) Case spouse (n = 30) Control (n = 28) Control spouse (n = 30)
67.8 30.0 16.7 10.0
100 100 100 100
82 66.7 26.7 26.7
100 100 100 100
*
κ statistics k = 1, P < 0.001.
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Table 3 Detection of HPV DNA in both urine and cervical/penile scrapes or tissue biopsies of women with cervical cancer and controls and their male sexual partners Study group
Total HPV positivity
Positive for HPV 16/18
Scrape/tissue (n = 30)
Urine
Women with cervical cancer Healthy women
25 (83.3%) 8 (26.7%) P ≤ 0.001*
Male partner to women with cervical cancer Male partner to healthy women
20 (66.7%) 8 (26.7%) P ≤ 0.005*
(n = 28)#
Scrape/tissue (n = 30)
Urine (n = 28)
23 (82%) 8 (26.7%) P ≤ 0.001*
21 (70%) 5 (16.7%) P ≤ 0.001*
19 (67.8%) 5 (16.7%) P ≤ 0.001*
20 (66.7%) 8 (26.7%) P ≤ 0.005*
9 (30%) 3 (10%) NS
9 (30%) 3 (10%) NS
NS = non-significant. # In two cases DNA was inadequate or absent hence no PCR could be done. * P value is significant when cases were compared with controls.
positivity and negativity (see Tables 2 and 3) between the two biological specimens. The frequency of HPV infection in cervical cancer cases and controls observed in the present study is in good agreement with earlier reports (Das et al., 1992a,b; Gopalkrishna et al., 1992). In the present study significantly higher frequency of HPV positivity (66.7%) was observed in male partners of women with invasive cervical cancer as compared to normal healthy controls (26.7%). Brinton and co-authors reported a low 23% prevalence of HPV among the 204 male partners of women with cervical cancer as compared to only 19.8% in 485 controls (Brinton et al., 1989). However, they used Filter in situ hybridization, which has a much lower sensitivity than PCR. Even using PCR, Castellsague and co-authors (1997) reported overall HPV prevalence of 17.5% only in male partners of women with cervical cancer, and as low as 3.5% in controls. A slightly higher rate of 25.7% HPV for male partners has been reported from Colombia (Munoz et al., 1996) and the difference was suggested to be due to higher prevalence of cervical cancer there. Recently, in a meta-analysis, Franceschi and co-authors also have reported a lower HPV prevalence of 18% among male partners of women with cervical cancer and 13% in controls (Franceschi et al., 2002). Certainly, in the present study, HPV detection rate either in urine or in scrape of male partners is significantly higher (66.7%). Furthermore, similar to present observations, studies world over have also recorded HPV type 16 as the most prevalent type in women with cervical cancer and in their male partners. Non-specific concordance rate for HPV infection among couples was found to be 76% in the study group as compared to 50% in control group. The type-specific concordance rate for high risk HPV DNA was 43% in study group as compared to 20% in controls. These findings are in good agreement with earlier reports (Baken et al., 1995; Strand et al., 1995). Some authors (Castellsague et al., 1997) who had reported lower concordance rates suggested that the specimens were not tested under identical conditions and for a number of couples, the sampling might have been done in irrelevant partners. It is interesting to note that the husbands of women with cervical cancer showed a significantly higher smoking habits and premarital or extramarital sexual relationships. Also the
mean age of marriage as well as the age of first coitus is significantly lower in those women who developed cancer (Table 1). In the present study, detection of HPV has been carried out simultaneously in urine and cervical scrape/tissue specimen in all subjects and no difference in HPV positivity/negativity between the two types of specimens could be observed. There is 100% concordance between urine and cervical scrape or biopsy. It demonstrates that PCR detection of HPV DNA in urine samples is a unique, non-invasive, and convenient method, which can be reliably employed for large-scale population screening, including women with low grade lesions (LSIL) or ASCUS/AGUS, a condition where conventional cytological Pap test failed to give diagnosis. This can be also used for studying mechanism of virus transmission. An easy access to urine facilitates its collection with less resistance than a Pap smear and allow screening larger number of individuals. Furthermore, urine sampling particularly selfcollection of urine (Prusty et al., 2005) can be specifically used for screening anogenital HPV infections in infants and adolescents in whom it is difficult to obtain genital scrapes or other specimens. This validation of urine sampling for reliable detection of HPV infection would serve as a most useful alternative approach for HPV vaccination monitoring. Although recently, there have been several reports regarding HPV detection in urine (Forslund et al., 1993; Geddy et al., 1993; Lazcano-Ponce et al., 2001a,b; Melchers et al., 1989; Nakazawa et al., 1991; Prusty et al., 2005; Tamin et al., 2002), the results do not show a good correlation between biopsy/scrapes and urine sampling. Furthermore, there is a very low detection rate of HPV infection in male partners of women with cervical cancer and HPV infection. Yet some authors even failed to detect HPV DNA in urine samples of male or female (Geddy et al., 1993; Lazcano-Ponce et al., 2001a,b). However, several reports including this report do suggest use of urine as an important non-invasive method (Forslund et al., 1993; Melchers et al., 1989; Nakazawa et al., 1991; Powell et al., 2003; Prusty et al., 2005; Tamin et al., 2002), which can be reliably employed for HPV DNA testing in cervical cancer screening programme. Besides HPV, urine sampling has recently been successfully used for detection of other urinogenital infections such as Chlamydia trachoma-
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tis, Gonorhoea, HIV (Brinkman et al., 2002; Hillman et al., 1993a; Tamin et al., 2002). The results, however, may differ due to different sample collection, DNA extraction and PCR protocol employed by different laboratories. Large population-based studies are needed for clinical application of this urine-based HPV DNA testing for screening genital infection in humans. Acknowledgement Financial assistance from Department of Biotechnology and ICMR is gratefully acknowledged. Authors also thank Dr. L. Satyanarayan for his statistical advice. References Baken LA, Koutsky LA, Kuypers J, Kosorok MR, Lee SK, Kiviat NB, et al. Genital human papillomavirus infection among male and female sex partners: prevalence and type-specific concordance. J Infect Dis 1995;171:429–32. Brinkman JA, Jones WE, Gaffga AM, Sanders JA, Chaturvedi AK, Slavinsky III J, et al. Detection of human papailloamvirus DNA in urine specimens from human immuno deficiency virus-positive women. J Clin Microbiol 2002;40:3155–61. Brinkman JA, Rahmani MZ, Jones WE, Chaturvedi AE, Hagensee ME. Optimization of PCR based detection of human papillomavirus DNA from urine specimens. J Clin Virol 2004;29:230–40. Brinton LA, Reeves WC, Brenes MM, Herrero R, Gaitan E, Tenorio F, et al. The male factor in the etiology of cervical cancer among sexually monogamous women. Int J Cancer 1989;44:199–203. Castellsague X, Ghaffari A, Daniel RW, Bosch FX, Munoz N, Shah KV. Prevalence of penile human papillomavirus DNA in husbands of women with and without cervical neoplasia: a study in Spain and Columbia. J Infect Dis 1997;176:353–61. Das BC, Gopalkrishna V, Hedau S, Katiyar S. Cancer of uterine cervix and human papillomavirus infection. Curr Sci 2000;78:52–63. Das BC, Gopalkrishna V, Sharma JK, Roy M, Luthra UK. Human papillomavirus DNA in urine of women with preneoplastic and neoplastic cervical lesions. Lancet 1992a;340:1417. Das BC, Sharma JK, Gopalkrishna V, Das DK, Singh V, Gissmann L, et al. A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by southern blot hybridization and polymerase chain reaction. J Med Virol 1992b;36:239–45. Fife KH, Coplan PM, Jansen KU, Dicello AC, Brown DR, Rojas C, et al. Poor sensitivity of polymerase chain reaction assays of genital skin swabs and urine to detect HPV 6 and 11 DNA in men. Sex Trans Dis 2003;30:246–8. Forslund O, Hansson BG, Rymark P, Bjerre B. Human papillomavirus DNA in urine samples compared with that in simultaneously collected urethra and cervix samples. Sweden J Clin Microbiol 1993;31:975–9. Franceschi S, Castellsague X, Dal Maso L, Smith JS, Plummer M, Ngelangel C, et al. Prevalence and determinants of human papillomavirus genital infection in men. Br J Cancer 2002;86:705–11. 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;107:127–33. Franceschi S, Rajkumar R, Snijders PJ, Arslan A, Mahe C, Plummer M, et al. Papillomavirus infection in rural women in southern India. Br J Cancer 2005;92:601–6.
Geddy PM, Wells M, Lacey CJ. Lack of detection of human papillomavirus DNA in male urine samples. Genitourin Med 1993;69:276–9. Gopalkrishna V, Francis A, Sharma JK, Das BC. A simple and rapid method of high quantity DNA isolation from cervical scrapes for detection of human papillomavirus infection. J Virol Meth 1992;36:63–72. Hillman RJ, Botcherby M, Ryait BK, Hanna N, Taylor-Robinson D. Detection of human papillomavirus DNA in the urogenital tracts of men with anogenital worts. Sex Trans Dis 1993a;20:21–7. Hillman RJ, Ryait BK, Botcherby M, Taylor-Robinson D. Human papillomavirus DNA in the urogenital tracts of men with Gonorrhoea, penile warts or genital dermatoses. Genitourin Med 1993b;69:187–92. Lazcano-Ponce E, Herrero R, Munoz N, Hernandez-Avila M, Salmeron J, Levya A, et al. High prevalence of human papillomavirus infection in Mexican males: comparative study of penile-urethral swabs and urine samples. Sex Trans Dis 2001a;28:277–80. Lazcano-Ponce E, Herrero R, Munoz N, Cruz A, Shah KV, Alonso P, et al. Epidemiology of HPV infection among Mexican women with normal cervical cytology. Int J Cancer 2001b;91:412–20. Melchers WJ, Schift R, Stolz E, Lindeman J, Quint WG. Human papillomavirus detection in urine samples from male patients by the polymerase chain reaction. J Clin Microbiol 1989;27:1711–4. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. International Agency for Research on Cancer Multicenter Cervical Cancer Study Group Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348: 518–27. Munoz N, Castellsague X, Bosch FX, Tafur L, de Sanjosa S, Aristizabal N, et al. Difficulty in elucidating the male role in cervical cancer in Colombia, a high-risk area of the disease. J Natl Cancer Inst 1996;88:1068–75. Nakazawa A, Inoue M, Fujita M, Tanizawa O, Hakura A. Detection of human papillomavirus type 16 in sexual partners of patients having cervical cancer by polymerase chain reaction. Jpn J Cancer Res 1991;82:1187–90. Palefsky JM, Barrasso R. HPV infection and disease in men. Obst Gynecol Clin North Am 1996;23:895–916. Powell J, Strauss S, Gray J, Wojnarowska F. Genital carriage of human papillomavirus (HPV) DNA in prepubertal girls with and without vulval disease. Pediatr Dermatol 2003;20:191–4. Prusty BK, Kumar A, Arora R, Batra S, Das BC. Human papillomavirus DNA detection in self-collected urine. Int J Gynecol Obs 2005;90(3):223–7. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer directed amplification of DNA with a thermo-stable DNA polymerase. Science 1988;239:487–91. 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. Stanczuk GA, Kay P, Allan B, Chirara M, Tswana SA, Bergstrom S, et al. Detection of human papillomavirus in urine and cervical swabs from patients with invasive cervical cancer. J Med Virol 2003;71:110–4. Strand A, Rylander E, Wilander E, Zehbe I. Human papillomavirus infection in male partners of women with squamous intraepithelial neoplasia and/or high risk HPV. Acta Derm-Venereol (Stockh) 1995;75:312–6. Tamin H, Finan RR, Sharida HE, Rashid M, Almawi WY. Cervico vaginal coinfections with human papillomavirus and Chlamydia trachomatis. Diagn Microbiol Infect Dis 2002;43:277–81. Vizcano AP, Moreno V, Bosch FX, Munoz N, Barros-Dios XM, Borras J, et al. International trends in the incidence of cervical cancer: II squamous cell carcinoma. Int J Cancer 2000;86, 429–5. WHO. Control of cancer cervix. A WHO meeting. Bull WHO 1986; 64: 607–618. Zur Hausen H. Human papillomavirus and their possible role in squamous cell carcinoma. Curr Top Microbiol Immunol 1979;78:1–30.
Human papillomavirus DNA in urine samples of women with or without cervical cancer and their male partners compared with simultaneously collected cervical/penile smear or biopsy specimens Amita Gupta a , Raksha Arora a , Sanjay Gupta b , Bhupesh K. Prusty c , Uma Kailash c , Swaraj Batra a , Bhudev C. Das c,∗ a Department of Gynecology and Obstetrics, Maulana Azad Medical College, Lok Nayak Hospital, New Delhi Division of Cytopathology, Institute of Cytology and Preventive Oncology (ICMR), I-7, Sector-39, Noida 201301, UP, India Division of Molecular Oncology, Institute of Cytology and Preventive Oncology (ICMR), I-7, Sector-39, Noida 201301, UP, India b
c
Received 16 December 2005; received in revised form 20 April 2006; accepted 19 July 2006
Abstract Infection of specific types of high-risk human papillomaviruses (HPVs) causes cervical cancer in women. Conventional test for genital HPV infection requires collection of scraped cervical cells or biopsy specimens, which involves invasive procedures. Utility of non-invasive urine sampling for detection of HPV in women and their male sexual partners is controversial. The validation of this urine-based HPV DNA test is of immense value not only in screening large population and children but also for HPV vaccine monitoring in adolescents. We examined the frequency of high risk HPV types 16 and 18 in simultaneously collected urine samples and cervical scrapes or biopsy specimens from women with cervical cancer and their single lifetime male sexual partners in order to validate the utility of urine sampling as a reliable non-invasive method for detection of genital HPV infection. Thirty women with invasive cervical cancer and their husbands along with 30 age-matched normal healthy women including their husbands were recruited for the study. Cervical biopsies/scrapes from women subjects and penile scrapes from their husbands and urine samples from all of them were collected before taking biopsy or scrapes. HPV-L1 consensus primer as well as high-risk HPV (HPV 16 and 18) type-specific oligo-primers were used for PCR detection of HPV DNA. The total frequency of HPV in women with cervical cancer was found to be 83% (25/30) while it was only 67% (20/30) in their male partners but there was virtually no difference in results between urine and scrape or tissue biopsy either in women or their male partners. Although healthy women and their husbands showed similar frequency of HPV infection both in urine and scrape samples, there was a significant difference (p = 0.05) in the prevalence of high risk HPV type 16 in women with cervical cancer (70%) and their male partners (30%). Similar was the trend between control women and their male partners. The results also showed a very high prevalence of HPV type 16 among Indian women with cervical cancer while its frequency was significantly low in their single lifetime male partners. The case by case matching of HPV positivity and negativity between urine and cervical/penile scrapes or biopsies obtained from women and their male partners demonstrated that the non-invasive urine sampling can be reliably used for screening genital HPV infection in both men and women. © 2006 Published by Elsevier B.V. Keywords: Human papillomavirus; Cervical cancer; Women male partner; Urine; Cervical scrapes; PCR
1. Introduction Cervical cancer is the most common cancer in Indian women with an annual incidence of about 120,000 new cases, constituting almost 16% of world’s annual incidence ∗
Corresponding author. Tel.: +91 120 2575838; fax: +91 120 2579473. E-mail address: [email protected] (B.C. Das).
1386-6532/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jcv.2006.07.007
(WHO, 1986). Several epidemiological risk factors are associated with the development of this cancer including multiple sexual partners, sexual intercourse at an early age, number of pregnancies, poor genital hygiene, smoking, oral contraceptives etc. Specific types of high risk-human papillomaviruses (HR-HPVs) have been strongly implicated as principal causal agents in the development of cervical cancer (Das et al., 1992a, 2000; Vizcano et al., 2000; Zur
A. Gupta et al. / Journal of Clinical Virology 37 (2006) 190–194
Hausen, 1979; Munoz et al., 2003). Of more than 100 HPV types thus far described, about 30 types are associated with anogenital cancers and HPV types 16 and 18 are considered to be the major high-risk HPV (HR-HPV) types. In India, as high as 98% cervical cancer cases show presence of HPV as compared to 5–20% in normal healthy controls (Das et al., 2000). HPV 16 is the most prevalent type (90%) while a very low frequency (3%) of HPV type 18 is found in India (Das et al., 1992a, 2000). Recent reports also indicate a low prevalence of other high-risk HPV types in India (Franceschi et al., 2003, 2005; Sowjanya et al., 2005). As with all other sexually transmitted diseases, the approach to control and treat HPV-associated diseases should include assessment of both the index case as well as his or her sexual partner(s). Therefore, understanding manifestations, diagnosis, and treatment of HPV-related diseases of male genital tract is an important part of caring women with genital HPV infection. Thus, screening of male sexual partners for HPV infection has been suggested for women presenting with cervical squamous intraepithelial lesions (Palefsky and Barrasso, 1996). Despite strong evidence implicating specific HR-HPV types in cervical carcinogenesis and an apparent role of male partners in causation of this disease, genital HPV infection in men has not been thoroughly investigated. In particular, the male reservoir for HPV types detected in cervical neoplasia is poorly understood. Studies using PCRbased HPV detection among male partners of women with cervical neoplasia are few and none from India where cervical cancer and infection of HPV are highest in the world and it forms a major public health problem. The present study has therefore been designed to investigate the prevalence of high risk HPV type 16 and HPV type 18 in women with cervical cancer and their male sexual partners in comparison to normal healthy women and their husbands. Conventional test for genital HPV infection requires collection of scraped cervical cells or biopsy specimens, which involves invasive procedures in a clinic. Such invasive methods are not feasible for a large-scale population screening. Thus there is a need for a simple, reliable and a non-invasive method for screening general population. Urine sampling has been utilized for detection of various genital infections (Hillman et al., 1993a; Tamin et al., 2002) including HPV infection (Brinkman et al., 2002, 2004; Das et al., 1992b; Gopalkrishna et al., 1992; Hillman et al., 1993a,b; Stanczuk et al., 2003). However, utility of urine sampling for detection of HPV infection is controversial (Powell et al., 2003; Fife et al., 2003; Lazcano-Ponce et al., 2001a,b). We have therefore compared the frequency of HPV infection between cervical and penile scrapes or tumor biopsies and urine samples of both partners to validate the utility of urine sampling as a reliable non-invasive method for detection of HPV infection. 2. Materials and methods The study group comprised 30 women with histologically confirmed invasive cervical cancer and their husbands and the
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control group consisted of 30 age-matched (±5 years) women with normal or inflammatory or negative cervical cytology and their husbands. The women having multiple male sexual partners were excluded from the study. Informed consent was taken from all enrolled subjects and Institutional Ethical Committee approved using part of human materials obtained for routine diagnostic purposes for research investigation. All male partners were interviewed regarding their sexual behavior by male interviewers using a structured questionnaire. Cervical biopsies from women with cervical cancer and scraped cervical cells from normal healthy women were collected. Cervical cells were collected by scraping the ectocervix or the surface of cervical portio with a wooden Ayre’s spatula. The spatula along with cervical materials were transferred to collection bottles containing phosphate buffered saline (PBS) and stored at −70 ◦ C deep freezer till further processing. The penile cell samples were collected by swabbing the intrameatal and distal urethra, the external surface of the glans and the coronal sulcus of the penis with wet thin cotton-tipped swabs. A smear on glass slide was also prepared for Pap test and remaining cells were eluted in PBS and stored at −70 ◦ C. Urine samples were collected from both women and their male partners before taking biopsy or cervical scrape or penile swab. DNA extraction was done by using both organic and non-organic methods (Gopalkrishna et al., 1992). The PCR amplification of HPV-DNA sequences was carried out first by L1 consensus primers and then by HPV types 16 and 18specific primers using the method of Saiki et al. with some modifications (Saiki et al., 1988). The primers used were: (HPV16 upstream 5 -AAG GCC AAC TAA ATG TCA C-3 , HPV16 downstream 5 -CTG CTT TTA TAC TAA CCG G-3 ; HPV18 upstream 5 -ACC TTA ATG AAA AAC CAC GA3 , HPV18 downstream 5 -CGT CGT TTA GAG TCG TTC CTG-3 ). All the samples were initially screened by the L1 consensus primers (L1 upstream 5 -GCM CAG GGW CAT AAY AAT GG-3 , L1 downstream 5 -CGT CCM AAR GGA WAC TGA TC-3 , where, M = A or C, R = A or G, W = A or T, Y = C or T) with beta globin gene (5 -GAA GAG CCA AGG ACA GGT AC-3 and 5 -CAA CTT CAT CCA CGT TAC ACC-3 ) as an internal control. Statistical significance was calculated using χ2 as well as κ statistics where k = 1, p < 0.001.
3. Results Comparison of various epidemiological characteristics of cervical cancer cases and their male partners along with controls is presented in Table 1. The mean age of women with cancer was 41.7 years, while 42.1 years was for the control group. The mean age of husbands of women in the study group was 46.4 years, and that of the control group was 46.9 years (see Table 1). Substantial difference was also observed in sexual behavior of male partners of cancer cases when compared to that of controls. They reported significantly a higher
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Table 1 Comparison of various epidemiological characteristics in cervical cancer cases, controls and their partners
Age mean (S.D.) Age at marriage (S.D.) Age at first coitus (S.D.) Illiterate Smoke Husband illiterate Husband smoking Husband PMSC Husband extra-MSC Circumcised
Case (n = 30) Control (n = 30) OR
P value
41.7 (6.9) 16.8 (2.3) 17.2 (2.0) 70% 50% 46.7% 86.7% 26.7% 53.3% 6.7%
0.73 <0.001 <0.001 0.004 <0.001 0.028 <0.001 0.54 <0.01 0.39
42.1 (5.9) 21.0 (2.2) 21.1 (2.2) 33.3% 10% 20% 43.3% 20% 20% 13.3%
– – – 4.7 9.0 3.5 8.5 1.4 4.6 0.46
PMSC: pre-marital sexual contacts; extra MSC: extra marital sexual contacts; OR: odds ratio.
weekly frequency of sexual intercourse, extra-marital sexual contact, contact with prostitutes and are nonuser of condoms. A majority of women with cervical cancer and their husbands were smokers, illiterate and had recorded poor genital hygiene when compared to those of healthy control women and their male partners. The frequency of circumcision in partners of cancer patients was also very low in comparison to their normal counterpart (Table 1). Out of 30 women with cervical cancer, 9 had well differentiated, 16 moderately differentiated and 4 poorly differentiated squamous cell carcinoma (SCC), and one adenocarcinoma. Of 30 controls, 15 women had cytologically negative Pap smear and 15 had negative Pap smear with inflammation. All the penile scrapes (30 each) from male partners of cases as well as controls were found to be benign anucleate or nucleate squamous cells and no cytological evidence of HPV infection could be discerned. The frequency of total HPV infection as revealed by L1 consensus primer in women with invasive cervical cancer was found to be 83.3% while it was 82.1% in the urine samples from them. DNA could not be isolated in two urine samples. Barring these two cases, there is 100% concordance for the HPV positivity and negativity between urine and cervical scrape/biopsy (Table 2). HPV 16 was the most predominant type with a prevalence of 70% in cervical biopsy specimens and 68% in urine samples by PCR, as compared to only 16.7% in both types of controls. In control group, HPV DNA was detected in the same 8 (26.7%) women both in their cervical scrapes and urine samples, demonstrating 100% agreement between them (Table 2). Four of these eight women had normal or nega-
tive Pap smear while other four had negative Pap smear with inflammation. HPV type 16 was detected in five of them in both cervical scrapes and in urine, giving prevalence of 16.7% in each. HPV 18 was not detected in any of the cervical smear or urine samples of the control group. In case of male partners of women with cervical cancer, HPV DNA detection rate was 66.7% as compared to 26.7% (Table 3) in control males and the difference was found to be statistically significant (p < 0.001). Although the prevalence of HPV was lower in male partners, 100% matching was found in HPV positivity between scraped penile cells and urine samples and HPV 16 was also the predominant type detected among the male partners of women. Concordance between the two types biological specimens i.e. cervical/penile scrapes and urine samples was estimated considering only those samples for which the results were available for both the specimens. It was found that out of 28 samples (two samples being inadequate in urine), 23 were positive for HPV infections and the rest were negative. The results of urine samples revealed positivity for all these 23 cases that also showed positivity in their scrape material. Thus the total concordance of the positivity and negativity was found to be 100% for the total HPV infections. Similar concordance was found for the high-risk HPV type 16 infections. Out of 25 HPV positive women with cervical cancer, 19 of their male partners were HPV positive resulting in non-specific concordance rate for HPV infection as 76%. In control group, out of eight HPV positive women, four of their male partners were HPV positive, giving a non-specific concordance rate of 50%. Type-specific concordance for HPV type 16 was found to be 43% in study group as nine male partners of 21 HPV-16 positive women were found to be HPV 16 positive. In control group only one male partner out of five HPV 16 positive women was found to be positive for HPV 16, resulting in type specific concordance rate of 20% for HPV 16. The difference, however, was not found to be statistically significant.
4. Discussion In the present study, the results show virtually no difference in the frequency of HPV infection between cervical scrapes/biopsies and urine samples either in women or in their male partners leading to 100% agreement for the HPV
Table 2 Percentage of HPV positivity in urine samples and its agreement with scrape or tissue samples of cervical cancers and controls along with their spouse Type of subjects
% of HPV positivity
*%
of agreement with scrape/tissues
% of total HPV positivity
* % of agreement with scrape/tissues
Cases (n = 28) Case spouse (n = 30) Control (n = 28) Control spouse (n = 30)
67.8 30.0 16.7 10.0
100 100 100 100
82 66.7 26.7 26.7
100 100 100 100
*
κ statistics k = 1, P < 0.001.
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Table 3 Detection of HPV DNA in both urine and cervical/penile scrapes or tissue biopsies of women with cervical cancer and controls and their male sexual partners Study group
Total HPV positivity
Positive for HPV 16/18
Scrape/tissue (n = 30)
Urine
Women with cervical cancer Healthy women
25 (83.3%) 8 (26.7%) P ≤ 0.001*
Male partner to women with cervical cancer Male partner to healthy women
20 (66.7%) 8 (26.7%) P ≤ 0.005*
(n = 28)#
Scrape/tissue (n = 30)
Urine (n = 28)
23 (82%) 8 (26.7%) P ≤ 0.001*
21 (70%) 5 (16.7%) P ≤ 0.001*
19 (67.8%) 5 (16.7%) P ≤ 0.001*
20 (66.7%) 8 (26.7%) P ≤ 0.005*
9 (30%) 3 (10%) NS
9 (30%) 3 (10%) NS
NS = non-significant. # In two cases DNA was inadequate or absent hence no PCR could be done. * P value is significant when cases were compared with controls.
positivity and negativity (see Tables 2 and 3) between the two biological specimens. The frequency of HPV infection in cervical cancer cases and controls observed in the present study is in good agreement with earlier reports (Das et al., 1992a,b; Gopalkrishna et al., 1992). In the present study significantly higher frequency of HPV positivity (66.7%) was observed in male partners of women with invasive cervical cancer as compared to normal healthy controls (26.7%). Brinton and co-authors reported a low 23% prevalence of HPV among the 204 male partners of women with cervical cancer as compared to only 19.8% in 485 controls (Brinton et al., 1989). However, they used Filter in situ hybridization, which has a much lower sensitivity than PCR. Even using PCR, Castellsague and co-authors (1997) reported overall HPV prevalence of 17.5% only in male partners of women with cervical cancer, and as low as 3.5% in controls. A slightly higher rate of 25.7% HPV for male partners has been reported from Colombia (Munoz et al., 1996) and the difference was suggested to be due to higher prevalence of cervical cancer there. Recently, in a meta-analysis, Franceschi and co-authors also have reported a lower HPV prevalence of 18% among male partners of women with cervical cancer and 13% in controls (Franceschi et al., 2002). Certainly, in the present study, HPV detection rate either in urine or in scrape of male partners is significantly higher (66.7%). Furthermore, similar to present observations, studies world over have also recorded HPV type 16 as the most prevalent type in women with cervical cancer and in their male partners. Non-specific concordance rate for HPV infection among couples was found to be 76% in the study group as compared to 50% in control group. The type-specific concordance rate for high risk HPV DNA was 43% in study group as compared to 20% in controls. These findings are in good agreement with earlier reports (Baken et al., 1995; Strand et al., 1995). Some authors (Castellsague et al., 1997) who had reported lower concordance rates suggested that the specimens were not tested under identical conditions and for a number of couples, the sampling might have been done in irrelevant partners. It is interesting to note that the husbands of women with cervical cancer showed a significantly higher smoking habits and premarital or extramarital sexual relationships. Also the
mean age of marriage as well as the age of first coitus is significantly lower in those women who developed cancer (Table 1). In the present study, detection of HPV has been carried out simultaneously in urine and cervical scrape/tissue specimen in all subjects and no difference in HPV positivity/negativity between the two types of specimens could be observed. There is 100% concordance between urine and cervical scrape or biopsy. It demonstrates that PCR detection of HPV DNA in urine samples is a unique, non-invasive, and convenient method, which can be reliably employed for large-scale population screening, including women with low grade lesions (LSIL) or ASCUS/AGUS, a condition where conventional cytological Pap test failed to give diagnosis. This can be also used for studying mechanism of virus transmission. An easy access to urine facilitates its collection with less resistance than a Pap smear and allow screening larger number of individuals. Furthermore, urine sampling particularly selfcollection of urine (Prusty et al., 2005) can be specifically used for screening anogenital HPV infections in infants and adolescents in whom it is difficult to obtain genital scrapes or other specimens. This validation of urine sampling for reliable detection of HPV infection would serve as a most useful alternative approach for HPV vaccination monitoring. Although recently, there have been several reports regarding HPV detection in urine (Forslund et al., 1993; Geddy et al., 1993; Lazcano-Ponce et al., 2001a,b; Melchers et al., 1989; Nakazawa et al., 1991; Prusty et al., 2005; Tamin et al., 2002), the results do not show a good correlation between biopsy/scrapes and urine sampling. Furthermore, there is a very low detection rate of HPV infection in male partners of women with cervical cancer and HPV infection. Yet some authors even failed to detect HPV DNA in urine samples of male or female (Geddy et al., 1993; Lazcano-Ponce et al., 2001a,b). However, several reports including this report do suggest use of urine as an important non-invasive method (Forslund et al., 1993; Melchers et al., 1989; Nakazawa et al., 1991; Powell et al., 2003; Prusty et al., 2005; Tamin et al., 2002), which can be reliably employed for HPV DNA testing in cervical cancer screening programme. Besides HPV, urine sampling has recently been successfully used for detection of other urinogenital infections such as Chlamydia trachoma-
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