Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2010  |  Volume : 53  |  Issue : 4  |  Page : 681--685

Prevalence of high-risk human papillomavirus types 16 and 18 in healthy women with cytologically negative pap smear in Iran


Akbar Safaei1, Mahsa Khanlari1, Moghdeh Momtahen2, Ahmad Monabati1, Minoo Robati2, Sedigheh Amooei2, Behnaz Valibeigi1, Negar Azarpira1,  
1 Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Obstetrics and Gynecology, Shiraz University of Medical Sciences, Shiraz, Iran

Correspondence Address:
Negar Azarpira
Organ Transplant Research Center, Department of Pathology, Nemazi Hospital, Shiraz University of Medical Sciences, Shiraz
Iran

Abstract

Background: Because human papillomavirus (HPV) is one of the causal factors in cervical cancer, understanding the epidemiology of this infection is an important step towards developing strategies for prevention. Materials and Methods: We evaluated the prevalence of high-risk human papillomavirus Types 16 and 18 in cervical samples from 402 healthy women with normal Pap smears by testing with type-specific primers in the polymerase chain reaction. Participants were seen at two gynecological clinics affiliated to the Shiraz University of Medical Sciences in Iran. Result: The prevalence of positive HPV findings was 5.5%; high-risk HPV human papillomavirus Type 16 prevalence was 2% and no patient harbored HPV-18. The prevalence of HPV was 4.5% in younger age group and gradually increased to 20% in the 4 th decade. Conclusion: The prevalence of high-risk HPV was highest in the youngest women and gradually decreased with age. Overall, the prevalence of HPV in our population is low.



How to cite this article:
Safaei A, Khanlari M, Momtahen M, Monabati A, Robati M, Amooei S, Valibeigi B, Azarpira N. Prevalence of high-risk human papillomavirus types 16 and 18 in healthy women with cytologically negative pap smear in Iran.Indian J Pathol Microbiol 2010;53:681-685


How to cite this URL:
Safaei A, Khanlari M, Momtahen M, Monabati A, Robati M, Amooei S, Valibeigi B, Azarpira N. Prevalence of high-risk human papillomavirus types 16 and 18 in healthy women with cytologically negative pap smear in Iran. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Nov 17 ];53:681-685
Available from: http://www.ijpmonline.org/text.asp?2010/53/4/681/72030


Full Text

 Introduction



Cervical cancer is the third most common malignancy of the female genital system, and is the second most common cancer in women. [1] Human papilloma virus (HPV) DNA is present in 80 to 90% of squamous cell carcinomas and adenocarcinomas worldwide, and is also found in cervical intraepithelial neoplasia (CIN). [2],[3],[4] The most prevalent HPV genotypes are 16, 18, 31 and 45, found in 49%, 12%, 5% and 8% of the cases, respectively. Among them HPV-16 and HPV-18 are the most commonly reported genotypes. Genotype 16 is more frequently seen in squamous cell carcinoma, whereas genotype 18 is more common in adenocarcinoma. [5] According to the International Agency for Research on Cancer (IARC), these two HPV types are classified as carcinogens in humans. [6]

The risk factors for HPV viral persistence and carcinoma in situ are classified as host factors, environmental/behavioral factors, and viral factors. Age is an obvious host factor. [7],[8] Environmental/behavioral risk factors include smoking, prolonged hormonal contraceptive use, multiparity, coinfection with Chlamydia trachomatis, non-use of condoms by partners, and nutritional factors. [9],[10],[11],[12] Viral risk factors for HPV that have been studied to date include viral type and variant, viral load, the effect of multiple concurrent HPV infections, and detection of HPV E6 and E7 transcripts. [12],[13] Each country needs to know the prevalence rate of HPV in order to define its strategies for cancer prevention and future vaccination. [13] The IARC coordinated a series of prevalence surveys of HPV infection and CIN lesions in countries with high and low incidences of cervical cancer, and investigated the extent of the differences in HPV between age groups. Unfortunately, Iran was not among the seven Asian countries that participated in this study. [14] Studies on the prevalence of HPV infection in Iran have focused mainly on carcinomas,and little information is currently available regarding women with normal cervical cytology. [15] Ghaffari et al. [16] performed a cervical cytological examination in 127 women in the area of Tehran, Iran. Normal cervical cytology was found in 77 women and the HPV genome was present in 10 of the 77 (13%). The most prevalent genotypes among the infected samples from women with normal and abnormal cytology were HPV-16 (76%), HPV-18 (13%) and HPV-11/6 (9%). [16]

A single cervical cytology test as a cancer screening tool is associated with a significant false-negative rate; therefore more sensitive HPV testing (around 95% sensitivity) is needed, along with screening for CIN to detect high-grade intraepithelial neoplasia (CIN 2/3). [1] HPV testing recently received Food and Drug Administration (FDA),(USA) approval as a primary screening test in women 30 years and older. [17] In this study, we describe the age-specific prevalence and determinants of HPV DNA detection in a group of women with normal cytology in southern Iran.

Only two HPV genotypes (16 and 18) were tested in our study as these genotypes are considered markers of high risk for cervical cancer.

 Materials and Methods



The study population comprised 402 women who had a Pap smear with normal cytologic findings but with inflammation. All women were attending their routine annual gynecologic check-up at the Motahari and Zeinabieie Gynecology clinics affiliated with the University of Medical Sciences, Shiraz, and were seen between July and December 2008. The Ethics Committee of Shiraz University of Medical Sciences approved this study. The patients were informed about the purpose of the study and written informed consent to take part was obtained from all of them.

Cytologic Material

Routine Pap smears were obtained with an Ayre spatula, rolled onto a microscope slide and spray-fixed, and the endocervical side of the spatula was swirled in 1 ml of sterile phosphate buffered saline to release the remaining cells for analysis of HPV DNA. The samples were transported and stored at -20 C prior to further analysis.

Cytologic Diagnosis

All Pap smears were analyzed in the two cytological laboratories normally used by the office-based pathologists. The laboratories were not informed of the study, and the slides and accompanying paperwork did not contain any information about the study. The smears were assessed according to the 2001 Bethesda cytologic classification. [17]

HPV Detection

Nucleic acids were extracted with the QIAamp DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Assays with PCR to detect 317-bp b-actin DNA were performed with forward primer 5-ATCATGTTTGAGACCTCCAA-3 and reverse primer 5-CATCTCTTGCTCGAAGTCCA-3 for each processed specimen to assess DNA integrity. A GP5+ /GP6+ primer set (Metabion International, Martinsried, Germany) was used to amplify a 140-bp fragment of the common region in all HPV with 5-TTTGTTACTGTGGTAGATACT-3 as the forward primer and 5-GAAAAATAAACTGTAAATCATATT-3 as the reverse primer. [15]

The 25 μl mixture for the PCR reaction included approximately 0.25 μg extracted DNA, 200 mM of each dNTP (Sinagene, Tehran, Iran), 1 U Taq DNA polymerase (Sinagene), 2.5 μl 10 PCR buffer, and 20 pmol of each primer (TIB Molbiol, Berlin, Germany). Thermocycler conditions were initial denaturation at 94 C for 3 min, followed by 45 cycles of denaturation at 94 ºC for 30 s, annealing at 50 ºC for 60 s, and extension at 72 C for 30 s, with a final extension at 72 C for 5 min. The PCR products were analyzed by electrophoresis on 2% agarose gel. A 100 bp-range molecular weight ladder and appropriate negative and positive controls were run simultaneously.

HPV Genotyping

The HPV-positive samples were further analyzed for HPV genotyping to search for genotypes 16 and 18. Forward primer 5-TCAAAAGCCACTGTGTCCTG-3 and reverse primer 5-CGTGTTCTTGATGATCTGCA-3 were used to amplify HPV-16 DNA, and forward primer 5-GACACATTGGAAAAACTA AC- 3 and reverse primer 5-TAG TGC CCA GCT ATG TTG TG -3. [15] were used for HPV 18. PCR reactions were carried out in a total volume of 25 μl containing 2.5 μl 10 PCR buffer, 2 mM MgCl 2 , 200 mM of each dNTP (Sinagene), 1 U Taq DNA polymerase (Sinagene, Iran), and 20 pmol of each primer (a mixture of HPV-16 and HPV-18-specific primers) (TIB Molbiol). Thermocycler conditions were initial denaturation at 94 C for three minutes followed by 30 cycles of denaturation at 94 C for one minute, annealing at 58 C for one minute, and extension at 72 C for one minute, with final extension at 72 C for five minutes. The amplified fragments were visualized by electrophoresis on 2% agarose gels and stained with ethidium bromide.

Statistical Analysis

Results were analyzed with the aid of SPSS software (Statistical Package for the Social Sciences, version 15, SSPS Inc, Chicago, IL, USA). Chi- square and Fisher exact tests performed comparison of the frequencies between groups. A level of P<0.05 was considered significant.

 Results



We studied a total of 402 cytologically negative specimens from women aged between 20 and 72 years (mean±SD: 35.2±5.2 years). Two cases were excluded because of a negative internal control. There were 22 HPV-positive cases (by PCR) in 400 women, giving an HPV prevalence of 5.5%. Of these, eight cases were positive for high-risk HPV, and all eight were infected with HPV genotype 16; none of them showed co-infection with genotype 18. The remaining 14 PCR-positive women showed negative PCR results for HPV genotypes 16 and 18.

According to our findings, the prevalence of HPV is 4.5% in women aged between 20-40 years, this gradually increased to 20% in the older age group (50-59). The prevalence of high-risk HPV (HPV 16) is around 2% and all of these cases are among the younger women [Table 1]. The prevalence of high-risk HPV cases decreased to zero in the older age groups [Table 1].{Table 1}

Mean parity was 2, and around 70% of the patients were 18 years or older at first coitus [Table 1]. Neither parity nor age at first coitus was found to be important risk factor. Almost half of the women (46%) had no history of contraception, and 3.8% of these (seven patients) were infected with HPV. The number of women using an IUD at the time of the study was too small to allow us to evaluate this method of contraception as a potential risk factor. Of the 34 women using oral contraceptives, three were found to be HPV-positive (8.8%).

Gynecological symptoms such as vaginal discharge or lower abdominal pain were found to be very common overall, but there was no correlation between these clinical symptoms and presence of high-risk viral infection [Table 2]. No suspicious clinical findings were found on speculum examination in patients positive for high-risk HPV.{Table 2}

Genotype 16 was responsible for eight cases of HPV infection (36%) out of the 22 samples positive for HPV DNA (by PCR); the HPV genotype was not determined as part of this study in the remaining 14 cases (64%).

 Discussion



Although cervical carcinoma is both preven table and curable, incidence rates are almost six-times greater in developing countries than developed nations. [18] The major reason for this difference seems to be the lack of proper screening programs in developing countries. A database on the epidemiological background of precancerous lesions of the cervix, with emphasis on HPV infection and other established risk factors is a basic requirement for such programs. Several studies have been completed in women with normal cervical cytology in different parts of the world, such as the USA-Mexico border, Costa Rica, the UK, Canada, Brazil, Greenland and Denmark, and the combined outcomes form an international database. [19],[20],[21],[22],[23],[24],[25]

According to worldwide epidemiological studies, the prevalence of high-risk HPV varies geographically. [2],[25],[26] Overall, HPV prevalence in women with normal cervical cytology is estimated at between 1.4% to 25.6%. [2] The IARC survey in Asia reported a prevalence of 9.6%, of which 5.4% were high-risk types (age-standardized prevalence). HPV-16 was twice as frequent as any other high-risk type in all regions. [9],[10]

According to these population-based data, the FDA (USA) has currently approved DNA tests for 13 high-risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) as an adjunct to primary cytologic screening. [24] The IARC systematic reviews Proyecto Epidemiolσgico Guanacaste and ASCUS/LSIL Triage Study (ALTS) suggested that only seven HPV types (type 16, 18, 33, 45, 31, 52 and 58) provide the best options for cervical cancer screening. [9],[10],[27] Aside from these virological and epidemiological challenges, formal cost-benefit analyses and societal preferences are important in selecting the HPV types to target in cancer prevention. [28]

We found HPV DNA in 5.5% of the cases, with 2% prevalence for high-risk HPV (type 16). This pattern was similar to that in low-prevalence areas. Overall HPV prevalence rates were actually higher in older women, with the highest prevalence in 50-59 year olds. In the present study, high-risk HPV positivity was strongly age dependent, and was only detected among the youngest women.

Smith et al. in a global review found that HPV infection prevalence decreased with increasing age from a peak prevalence in younger women (< or =25 years of age). [28] Inconsistent trends in HPV prevalence by age were noted in older women, with a decrease or plateau of HPV prevalence in older ages in most studies, whereas others showed an increase of HPV prevalence in older ages. HPV positivity increased slightly in older aged women in Senegal (over 45 years of age) and in South Africa (over 50 years of age). [29] One study of women in Genoa reported an HPV prevalence of 15% in women 44 years of age and younger and found the highest prevalence (19%) in women 60 years and older. [30] Smith et al. [28] noted similar trends of HPV 16 and/or 18 prevalence by age. In our study, HR HPVs showed decreased occurrence with increasing age. Therefore, it suggested that HPV infections at a young age is transient and is eradicated by the immune system. Elimination of the virus may explain why not all HPV-infected women develop squamous intraepithelial lesions or invasive cervical cancer. [31],[32] We found no association between age at first coitus and risk of HPV. Das et al. [33] reported that HPV infection in women younger than 18 years at first coitus increased the risk of cervical cancer 22-fold. In contrast, other authors found no association of HPV positivity with an early age of sexual exposure. [34]

About 46% of women in our study had no history of contraception, and about 9% of participants used oral contraceptives. None of the women we studied gave a history of multiple sexual partners. Neither contraception nor multiple sexual partners were associated with HPV infection in this study. However, other studies found that multiple sexual partners and long-term oral contraceptive use could be a risk factor for HPV infection. [33],[34] Among major life style variables, tobacco smoking was noted to be positively associated with cervical abnormalities. [35] This has been attributed to the reduction in the number of Langerhans cells in the cervical epithelium. Smoking is uncommon or denied among women in our community.

In conclusion, this study confirms the low prevalence of HPV in southeastern Iran. We hope that further larger scale studies with a wider range of genotype testing will present a clearer picture of the other HPV genotypes circulating in our region. These epidemiological data can improve strategies in the prevention and treatment of HPV-associated lesions.

 Acknowledgments



This research was supported by Vice Chancellor of Research, affiliated to Shiraz University of Medical Sciences. We thank K. Shashok, S. Griffin-Mason and P. Waltl (AuthorAID in the Eastern Mediterranean) for improving the use of English in the manuscript.[36]

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