Banghyun Lee 1, Hye-Yon Cho 2, Kum Jin Jeon 1, Kidong Kim 1,3, Jung Ryeol Lee 1,3, Jung Joo Moon 4, Jae Hong No 1,3 and Yong-Beom Kim 1,3.

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1 doi: /jog J. Obstet. Gynaecol. Res. Vol. 42, No. 3: , March 2016 Detection of high-risk human papillomavirus using menstrual blood in women with high-grade squamous intraepithelial lesions or high-risk human papillomavirus infections: A pilot study Banghyun Lee 1, Hye-Yon Cho 2, Kum Jin Jeon 1, Kidong Kim 1,3, Jung Ryeol Lee 1,3, Jung Joo Moon 4, Jae Hong No 1,3 and Yong-Beom Kim 1,3 1 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam-Si, 2 Department of Obstetrics and Gynecology, Hallym University Dongtan Sacred Heart Hospital, Hwaseong-Si, 4 Goodgene Inc. Koyang-Si, Gyeonggi-do, and 3 Seoul National University, School of Medicine, Seoul, Republic of Korea Abstract Aim: Few studies have reported that human papillomavirus (HPV) tests using menstrual blood (MB) may be a convenient and effective screening modality for cervical cancer. Therefore, we aimed to investigate the efficacy of detecting high-risk (HR)-HPV from MB in women with high-grade squamous intraepithelial lesions or HR-HPV infections dependent on menstrual days. Materials and Methods: In this prospective exploratory pilot study, a clinical trial was conducted in 19 women. On enrollment into the study, gynecologists collected cervical cells. On the first and second day of menstruation, MB was self-collected by patients using a sanitary pad with a filter. The distribution of HPVs from MB and the accuracy of menstrual HR-HPV tests were evaluated using HPV genotyping. The agreement rate of detecting HR-HPVs using cervical and MB samples was also investigated. Results: The sensitivity, specificity, positive and negative predictive values of the MB HR-HPV test for detecting cervical intraepithelial neoplasia (CIN) 3 or worse were 87.5%, 45.5%, 53.8%, and 83.3%, respectively, during both menstrual cycle day (MCD) 1 and 2 and MCD 1 only; and 62.5%, 27.3%, 38.5%, and 50.0%, respectively, during MCD 2 only. For CIN 3 or worse, the agreement rate between positive cervical and MB HR-HPV test results was 87.5% during MCD 1 and 62.5% during MCD 2. Conclusions: We demonstrated the possibility of using the MB HPV test as a screening modality for cervical cancer. Key words: cervical intraepithelial neoplasia, genotyping technique, high-grade squamous intraepithelial lesion, human papillomavirus, menstruation. Introduction Cervical cancer is the most commonly diagnosed gynecologic cancer worldwide, and the most common cause of gynecologic cancer death. 1 Human papillomavirus (HPV) is the leading cause of cervical cancer. Cervical cancer may be detected at early or precancerous stages by cervical cytology or HPV testing. 2 HPV testing is more sensitive than cytology alone for detecting cervical intraepithelial neoplasia (CIN) 2, CIN 3 or worse, and is Received: August Accepted: September Reprint request to: Kidong Kim, Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 82, Gumi-ro 173 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea. kidong.kim.md@gmail.com 2015 Japan Society of Obstetrics and Gynecology 319

2 B. Lee et al. as sensitive as co-testing using HPV and cytology. 3 However, the global rate of women receiving cervical cancer screening remains low despite overall increases in the screening rate. 4 Factors, such as embarrassment, fear of pain, difficulty making an appointment with the clinician and not finding time to visit the clinic, have been considered to be barriers in cervical cancer screening. 5 To improve screening coverage in women who do not participate in screening programs, patient-performed vaginal HPV tests using a brush, swab, lavage and tampon have been investigated. The self-hpv tests in a recent meta-analysis were highly sensitive (84%) and specific (87%) for CIN 3 or worse (76% and 86%, respectively, for CIN 2 or worse), but lower than clinician-performed cervical HPV tests. 6 Patient-performed HPV screening tests are inconvenient for collection, storage and delivery of specimens within liquid media. Only a few studies have reported the detection of HPVs in menstrual blood (MB) using a sanitary pad. 7 9 Pads can more conveniently be enclosed in a Ziploc bag and sent to the laboratory by mail. 7,8 MB HPV DNA was reported to have a sensitivity and specificity of 82.8% and 93.1%, respectively, for CIN or condyloma acuminatum. 8 In another study, MB HPV DNA was observed to have a sensitivity of 76.9% and specificity of 93.3% for CIN, which was similar to that forcin2orcin3. 9 These findings suggest that HPV tests using MB would be effective and provide more convenient screening compared to other patient-performed vaginal HPV tests. However, previous studies were limited by either using a case control study design, participants had undergone recent punch or cone biopsy, a low clinical association was attributed to detecting overall HPV DNA types without targeting high-risk (HR) types oralackofdatatodetectcin2orcin3wasfound. In order to evaluate whether HPVs are detected in MB, a study of women with cervical HPV infections is required. A majority of women with high-grade squamous intraepithelial lesion (HSIL) cytology are infected with cervical HR-HPV. 10 Therefore, we designed a prospective pilot study to determine the distribution of HR-HPV types and efficacy of HR-HPV tests using MB from women with HSIL cytology or cervical HR-HPV infections depending on menstrual cycle day (MCD). Furthermore, we wanted to examine the agreement rate of detecting HR-HPVs between MB and cervical samples and determine the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for detecting CIN 2 and 3 or cervical cancer. Data from this study may be used to provide valuable and necessary information for the future clinical use of MB HPV tests. Materials and Methods The Institutional Review Board of Seoul National University Bundang Hospital approved this study. Our prospective exploratory study included women who were enrolled in a clinical trial at the Seoul National University Bundang Hospital from 1 January to 30 June The inclusion criteria were as follows: women with HSIL cytology or positivity for HR-HPVs on the hybrid capture (HC) 2 HPV DNA test or HPV genotyping with screening performed within three months; and premenopausal women. The exclusion criteria were as follows: absence of a cervix (i.e. previous surgery); women who were recommended rapid treatment by their physician because of suspected cervical cancer; diagnosis of CIN; or women undergoing punch biopsy or conization within three months from the onset of the study. Written informed consent was obtained from all participants. Collection of specimens Cervical cells were collected by gynecologists from the participants during enrollment using an AMPLICOR STD Swab Specimen Collection and Transport Kit. MB was self-collected by the participants. On the first day of the first menstruation following enrollment, apadwithafilter was attached to the underwear so that the filter of the pad would lie directly below the vaginal orifice. After complete usage of the pad (e.g. collection of large amounts of MB), the pad filter was removed using clear gloves. The filter was contained in a clean tube and preserved in a refrigerator at 4 C. The process was repeated on the second day of menstruation. The participants were informed about the self-collection processes prior to initiating the study. The final pathologic diagnosis was conducted using punch biopsy, endocervical curettage, conization or hysterectomy. DNA extraction Total DNA from cervical cells was extracted using a Labopass Tissue Miniprep kit according to the manufacturer s instructions. A 1 cm 1 cm sized sample was cut from the participants pads using sterile scissors. The total DNA from each sample pad was extracted using a QIAamp DNA Mini kit or LaboPass Tissue Miniprep kit, according to the manufacturer s protocol. The total amount of DNA was quantified at a wavelength of 260 nm using spectrophotometry Japan Society of Obstetrics and Gynecology

3 Detection of HPV in menstrual blood Duplex polymerase chain reaction The primers used for amplification were as follows: L1 sequence 5 -GCMCAGGGW CATAAYAATGG-3 ;L2sequence 5 -AATAAACTGTAAATCATATTCCTC-3 ; GP6 sequence 5 -GAAAAATAAACTGTAAATCATATTC-3 ;andbeta-actin sequence, forward 5 -GCACCACACCTTCTACAATGA-3 and reverse 5 -GTCATCTTCTCGCGG TTGGC-3. The regions amplified with these primers were 185 bp and 102 bp for HPV and beta-actin, respectively. A polymerase chain reaction (PCR) amplification of a 7 μl DNA template was performed in a 30 μl reaction containing 1 μl each of L1, L2 and GP6+ primer and 15 μl premix using the GG HPV40 DNA chip PCR kit and a 2720 Thermal Cycler. The amplification conditions were: 95 C for five min, 40 cycles at 95 C for 30 s, 54 C for 30 s and 72 C for 30 s, followed by a final extension at 72 C for five min. The annealing temperature was 50 C for all primers. Caski and Hela cells were used as positive controls and human embryonic kidney cells 293 cells were used as negative controls. The PCR products were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. HPV genotyping The amplified products were mixed with a hybridization reaction buffer (provided by the manufacturer) for the hybridization reaction, which was performed in a chamber at 50 C for 30 min. The hybridized signal on the HPV DNA chip was visualized using a GenePix 4000B Microarray Scanner. The genotyping kit detected 40 HPV types: high-risk, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68a, 68b and 82; possible high-risk, HPV 26, 53, 66, 67, 69, 70 and 73; low-risk, HPV 6, 11, 34, 40, 42, 43, 44, 54, 55, 61, 62, 72, 81 and 90; and undetermined risk, HPV 30, 32, 83, 84 and 91. Efficacy assessments The age, MCD of MB collection, presence or absence of HPVs in the cervical cells or MB, types of HPVs detected in the cervical cells or MB and final diagnoses were analyzed. Participants who had provided cervical cell samples and >1 pad stained with MB were included in the analyses. Efficacy was assessed according to sensitivity, specificity, PPV and NPV and the following agreement rates: (i) agreement rate of HPV testing performed using MB based on participants = (participants with both positive cervical and MB HPV test results/all participants with positive cervical HPV test results) 100 (%); and (ii) agreement rate of HPV testing performed using MB based on HPV types = (participants with same HPV types detected by both cervical and MB HPV tests/all participants with positive cervical HPV test results) 100 (%). Agreement rates for MB HPV (i.e. those based on participants or on HPV types) of >80% were defined as significant agreement for clinical applications. Sample size Based on our clinical experience, approximately six premenopausal women visit our hospital per month to obtain further evaluation or treatment for their diagnosis of HSIL cytology or positive HR-HPVs. Therefore, with a consent rate of 50%, three premenopausal women per month were predicted to be included in this clinical trial. We had originally aimed to collect 20 participants for six months. A total of 19 women participated in our trial. Results The median age of the study population was 37 years (range: years) and the mean age was ± 6.35 years (mean ± standard error). Pap smear results varied in the study population. The distribution of HPV types and pathology in the study population are shown in Table 1. The participants had HPV 16, 18, 31, 33, 35, 56, 58, 66 and 68, with the exception of two patients who underwent HC 2. HPV 16, 18, 31, 33, 35, 54, 56, 58, 59, 66, 67, 68b, 70 and 82 were detected in the cervix. On MCD 1, HPV 16, 18, 31, 33, 35, 45, 52, 54, 56, 58, 66, 68b, 70 and 82 were detected in MB. On MCD 2, HPV 16, 18, 31, 33, 35, 45, 52, 54, 56, 58, 66, 70 and 82 were detected in MB. Final pathologic findings included: koilocytosis; CIN 1, 2, and 3; and cervical cancer 1A1. The HR-HPV tests, using both cervical and MB samples, were observed to have a high sensitivity and NPV, but low specificity and PPV for detecting CIN 2 and CIN 3 or worse. The sensitivity, specificity, PPV and NPV of the MB HR-HPV tests were as follows (respectively): for detecting CIN 3 or worse, 87.5%, 45.5%, 53.8% and 83.3% during MCD 1 and 2, 87.5%, 54.5%, 58.3% and 85.7% during MCD 1 only and 62.5%, 54.5%, 50.0% and 66.7% during MCD 2 only; for detecting CIN 2 or worse, 88.9%, 50.0%, 61.5% and 83.3% during MCD 1 and 2, 77.8%, 50.0%, 58.3% and 71.4% during MCD 1 only and 66.7%, 60.0%, 60.0% and 66.7% during MCD 2 only. The specificity and PPV were also similar between the cervical and MB HR-HPV tests. The sensitivity, specificity, PPV and NPV of the HR-HPVtestsusingcervicalsampleswereasfollows: 2015 Japan Society of Obstetrics and Gynecology 321

4 B. Lee et al. Table 1 Distribution of human papillomavirus types and pathology in the study population Participants Age Pap smear Prior HPV test Cervical MB HPV Pathology (n = 19) HPV MCD 1 MCD ASC-US Koilocytosis 2 32 ASC-H 16,58 16,58,82 16,58 16,58,82 CIN ASC-H 68 68b 68b 0 CIN LSIL HR 18 16,18 18 CIN HR 16 16,52 16,45,52 CIN HSIL 16 70,82 70,82 70 CIN HSIL CIN ASC-US Koilocytosis 9 37 HSIL ,31 CIN ASC-H CIN HSIL ,33 33 Cervical cancer 1A LSIL Koilocytosis ASC-US CIN ASC-US CIN HSIL 0 16,18,54,59, CIN ASC-US ,70 70 CIN WNL CIN WNL 16,35 16,35 16,18,35 16,18,35 Koilocytosis ASC-US Koilocytosis ASC-H, atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion; ASC-US, atypical cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; MB, menstrual blood; MCD, menstrual cycle day; WNL, within normal limits. Table 2 Accuracy of high-risk human papillomavirus tests performed using cervical and menstrual blood samples to detect cervical intraepithelial neoplasia 2, 3, or cervical cancer Cervix MB MCD 1 MCD 2 MCD 1 & 2 Sensitivity CIN2 or worse (n = 9) 100% (9/9) 77.8% (7/9) 66.7% (6/9) 88.9% (8/9) CIN3 or worse (n = 8) 100% (8/8) 87.5% (7/8) 62.5% (5/8) 87.5% (7/8) Specificity CIN2 or worse (n = 9) 50.0% (5/10) 50.0% (5/10) 60.0% (6/10) 50.0% (5/10) CIN3 or worse (n = 8) 45.5% (5/11) 54.5% (6/11) 54.5% (6/11) 45.5% (5/11) Positive predictive value CIN2 or worse (n = 9) 64.3% (9/14) 58.3% (7/12) 60.0% (6/10) 61.5% (8/13) CIN3 or worse (n = 8) 57.1% (8/14) 58.3% (7/12) 50.0% (5/10) 53.8% (7/13) Negative predictive value CIN2 or worse (n = 9) 100% (5/5) 71.4% (5/7) 66.7% (6/9) 83.3% (5/6) CIN3 or worse (n = 8) 100% (5/5) 85.7% (6/7) 66.7% (6/9) 83.3% (5/6) CIN, cervical intraepithelial neoplasia; MB, menstrual blood; MCD, menstrual cycle day. detecting CIN 3 or worse, 100.0%, 45.5%, 57.1% and 100.0%, respectively; and detecting CIN 2 or worse, 100.0%, 50.0%, 64.3% and 100.0%, respectively (Table 2). The agreement rate for patients with positive cervical HR-HPV test results having positive MB HR-HPV test results was 85.7% during MCD 1 and 2 (78.6% for MCD 1, 71.4% for MCD 2). The agreement rate of the MB HR-HPV test during MCD 1 and 2 was 87.5% (87.5% for MCD 1, 62.5% for MCD 2) for detecting CIN 3 or worse and 88.9% (77.8% during MCD 1, 66.7% during MCD 2) for detecting CIN 2 or worse. These findings matched the agreement rates for patients with positive cervical HR-HPVs having the same types of MB HR-HPVs (Table 3). Furthermore, the agreement rates were similar when other possible HR-HPV types were included in analyses (data not shown) Japan Society of Obstetrics and Gynecology

5 Detection of HPV in menstrual blood Table 3 Agreement rates, for detecting high-risk human papillomavirus in menstrual blood compared to cervical samples MB MCD 1 MCD 2 MCD 1 & 2 All (n = 14) 78.6% (11/14) 71.4% (10/14) 85.7% (12/14) CIN 2 or worse (n = 9) 77.8% (7/9) 66.7% (6/9) 88.9% (8/9) CIN 3 or worse (n = 8) 87.5% (7/8) 62.5% (5/8) 87.5% (7/8) Agreement rate of human papillomavirus (HPV) test performed using MB based on participants = (participants with both positive cervical and MB HPV test results/all participants with positive cervical HPV test results) 100 (%). Agreement rate of HPV test performed using MB based on HPV types = (participants with same HPV types detected by both cervical and MB HPV tests/all participants with positive cervical HPV test results) 100 (%). and were equal to each other. CIN, cervical intraepithelial neoplasia; MB, menstrual blood; MCD, menstrual cycle day. Discussion Previous studies have provided evidence to support the effectiveness of HPV testing as a modality for primary screening and secondary prevention of cervical cancer In the future, HPV testing alone is expected to be used as a useful method for the screening and triage of cervical cancer. 3,11 In this study, we evaluated the distribution of HR-HPVs in MB, which will be helpful for testing HPV alone or co-testing with cervical cytology. The presence of HR-HPVs was similar between the cervical and MB samples (Table 1). In a study that involved samples collected by vaginal tampons, the HR-HPV detection rates of vaginal discharge were high in participants with long tampon exposure (1 h, 4 h and overnight), but low in those with a 10 s tampon exposure. 12 Therefore, using a sanitary pad may provide the opportunity to detect MB HR-HPV, given a sufficient exposure time. The sensitivity and specificity of the MB HPV DNA test in a previous study were 75.3% and 96.6%, respectively, for detecting CIN 2 or CIN 3. 9 We were able to show that in a CIN-enriched population, the MB HR-HPV test had a higher sensitivity and lower specificity for detecting CIN 2 and CIN 3 or worse, compared with the results from this previous study, without targeting HR-HPV DNA. 9 In our study, the high sensitivity and NPV and low specificity and PPV for detecting CIN 2 and CIN 3 or worse were observed in both the cervical and MB HR-HPV tests. These findings partially corresponded to previous reports, in which the sensitivity, specificity, PPV and NPV were observed to be 69.2%, 63.2%, 30.0% and 90.0%, respectively, for detecting CIN 3, and a sensitivity of % and specificity of % for detecting CIN 3 or more for cervical HR-HPV DNA types. 13,14 In our study, the sensitivity and NPVof the MB HR-HPV tests were lower compared with the cervical HR-HPV tests, which corresponds to previous findings of lower sensitivity and specificity for detecting CIN 3 and CIN 2 or worse in self-hpv tests, compared with clinician-performed cervical HPV tests (Table 2). 6 According to the analysis of MB collected from the sanitary pads, MB HR-HPV DNA was detected in 89.8% of HPV DNA-positive women with CIN 2 and CIN 3 and 26.7% of HPV DNA-positive women with CIN 1 or condyloma acuminatum. 8 Additionally, in a study involving a general population of patients who visited clinics for routine examinations, the HPV tests using MB and clinician-performed cervical HPV tests had 97.8% agreement for the detection of HPVs. 9 Although previous investigators have reported a high agreement rate for detecting HR-HPVs from cervical and MB samples, they did not target HR-HPVs or the studies were performed in a case control setting. 7 9 In contrast to previous studies, we were able to provide data regarding the detection of HR-HPV in MB, especially in patients with CIN 2, 3 and cancer, according to MCD, in a prospective cohort setting (Table 1 and 3). The agreement rate for detecting HR-HPVs from cervical and MB samples was higher when the results from MCD 1 and 2 were combined compared with when MCD 1 and 2 were analyzed independently. Interestingly, we were able to show that the agreement rates were equal between women who had positive cervical and MB HR-HPV test results when analyzed based on participants or HPV types. These findings may be explained by the small sample sizes. Large-scale studies might be useful for clarifying these incidental findings (Table 3). In the current study, the sensitivity, specificity, PPV and NPVof the MB HR-HPV tests and the agreement rates for detecting HR-HPVs were higher during MCD 1 compared to MCD 2. According to these findings, the viral loads of HR-HPV infections might decrease according to the progression of menstruation. Moreover, we were able to observe that the sensitivity and NPV of MB HR-HPV tests and the agreement rates for detecting HR-HPVs using cervical and MB samples were highest when performed during MCD 1 in women with CIN 3 or worse Japan Society of Obstetrics and Gynecology 323

6 B. Lee et al. Based on the analysis including possible HR-HPV types, the highest agreement rate was 87.5% for detecting MB HR-HPVsonMCD1inwomenwithCIN3orworse. However, the agreement rate of detecting HR-HPVs using cervical and MB samples in women with CIN 2 or worse were similar to those with any pathology during their MCDs. Previous studies have reported that an elevation in cervical HPV-16 DNA load was associated with an increased risk of CIN 3. Furthermore, in women with higher HPV-16 loads, there was an increasing trend to progress toward a higher-grade CIN. 15,16 Therefore, we presumed that CIN 3 or worse with high cervical HR-HPV loads were associated with a higher presence of HR-HPV in MB compared to koilocytosis, CIN 1 and CIN 2 with relatively low cervical HR-HPV loads because the viral loads of HR-HPV in MB might be diluted according to the progression of menstruation. According to our study, the sensitivity, specificity, PPV and NPV of the MB HR-HPV test and the agreement rates between cervical and MB HR-HPV results were lowest when measured during MCD 2 in women with CIN 3 or worse. These findings may be explained by the small sample sizes, which is a limitation of this study. In summary, we conducted a prospective exploratory pilot study to evaluate the expression of MBHPVduringMCD1and2in19womenwith HSIL cytology or HR-HPV infection. We demonstrated that MB HR-HPV tests have high sensitivity and NPV, but low specificity and PPV for CIN 3 and CIN 2 or worse. Women with cervical HR-HPVs also had a high frequency of MB HR-HPV expression. The efficacy of the MB HR-HPV test for detecting HR-HPVs was highest during MCD 1 in women with CIN 3 or cancer. The MB HPV test provides a new screening modality that may significantly improve accessibility for cervical cancer screening. Large prospective randomized trials are needed to confirm efficacy and develop a standardized method for MB HPV testing. Acknowledgments This study was supported by grant no from the Seoul National University Bundang Hospital Research Fund. Conflict of interest The authors have no conflicts of interest relevant to this article. References 1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN Int J Cancer 2010; 127: Miller C, Elkas JC. Cervical and Vaginal Cancer. In: Berek JS (ed). Berek & Novak s Gynecology, 15th edn. Philadelphia: Lippincott Williams & Wilkins, Wolters Kluwer Health, 2012; Saslow D, Solomon D, Lawson HW et al. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. CA Cancer J Clin 2012; 62: Park MJ, Park EC, Choi KS, Jun JK, Lee HY. Sociodemographic gradients in breast and cervical cancer screening in Korea: The Korean National Cancer Screening Survey (KNCSS) BMC Cancer 2011; 11: Waller J, Bartoszek M, Marlow L, Wardle J. Barriers to cervical cancer screening attendance in England: A population-based survey. J Med Screen 2009; 16: ArbynM,VerdoodtF,SnijdersPJet al. Accuracy of human papillomavirus testing on self-collected versus clinician-collected samples: A meta-analysis. Lancet Oncol 2014; 15: Tong TR, Chan OW, Chow TC, Yu V, Leung KM, To SH. Detection of human papillomavirus in sanitary napkins: A new paradigm in cervical cancer screening. Diagn Cytopathol 2003; 28: Wong SC, Au TC, Chan SC et al. Human papillomavirus DNA detection in menstrual blood from patients with cervical intraepithelial neoplasia and condyloma acuminatum. JClin Microbiol 2010; 48: Kim SR, Song SY, Kim DS et al. Pad - a new self-collection device for human papillomavirus. Int J STD AIDS 2007; 18: Hwang HS, Park M, Lee SY, Kwon KH, Pang MG. Distribution and prevalence of human papillomavirus genotypes in routine pap smear of 2,470 Korean women determined by DNA chip. Cancer Epidemiol Biomarkers Prev 2004; 13: Arbyn M, Ronco G, Anttila A et al. Evidence regarding human papillomavirus testing in secondary prevention of cervical cancer. Vaccine 2012; 30 (Suppl 5): F88 F Harper DM, Raymond M, Noll WW, Belloni DR, Duncan LT, Cole BF. Tampon samplings with longer cervicovaginal cell exposures are equivalent to two consecutive swabs for the detection of high-risk human papillomavirus. Sex Transm Dis 2002; 29: Pajtler M, Milicic-Juhas V, Milojkovic M, Topolovec Z, Curzik D, Mihaljevic I. Assessment of HPV DNA test value in management women with cytological findings of ASC-US, CIN1 and CIN2. Coll Antropol 2010; 34: Kulasingam SL, Hughes JP, Kiviat NB et al. Evaluation of human papillomavirus testing in primary screening for cervical abnormalities: Comparison of sensitivity, specificity, and frequency of referral. JAMA 2002; 288: Xi LF, Hughes JP, Castle PE et al. Viral load in the natural history of human papillomavirus type 16 infection: A nested case control study. J Infect Dis 2011; 203: Fiander AN, Hart KW, Hibbitts SJ et al. Variation in human papillomavirus type-16 viral load within different histological grades of cervical neoplasia. JMedVirol2007; 79: Japan Society of Obstetrics and Gynecology