Clinical validation of high risk HPV DNA testing versus ThinPrep cytology for primary cervical cancer screening

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1 Middle East Fertility Society Journal (2013) 18, Middle East Fertility Society Middle East Fertility Society Journal ORIGINAL ARTICLE Clinical validation of high risk HPV DNA testing versus ThinPrep cytology for primary cervical cancer screening Mahmoud El-Morsi Aboul-Fotouh a,b, *,1, Ihab Talaat Hana c,d a OB/GYN Dept., Faculty of Medicine, Minia University, Minia, Egypt b OB/GYN Dept., Taiba Hospital, Kuwait c Assistant Professor of Pathology, Faculty of Medicine, Minia University, Minia, Egypt d Consultant Pathologist, Taiba Hospital, Kuwait Received 2 December 2012; accepted 9 December 2012 Available online 18 January 2013 KEYWORDS HPV; HPV DNA testing; Pap smear; ThinPrep cytology; Cervical cancer Abstract Objective(s): To compare the validity of the high risk HPV DNA testing using the hybrid capture II technique (HC-II) to ThinPrep cytology for primary cervical cancer screening. Design: Cross sectional pilot study. Setting: Department of Obstetrics and Gynecology, Taiba Hospital, Sabah Al Salem, Kuwait. Methods: Consecutive 1923 cervical smear samples were taken for ThinPrep cytological screening and hr-hpv DNA testing using HC-II assay. Histological diagnoses were obtained from a total of 426 women who had positive results on screening and a group of women with negative screening and suspicious cervix underwent colposcopy and directed biopsies, and those with cervical precancerous lesions or cancer received appropriate treatment. Main outcome measures: Sensitivity, specificity, positive predictive value and negative predictive value of screening methods. Results: HPV was found positive in 15.5% of cases. 19/22 cases (86.4.1%) with a biopsy diagnosis of CIN2+ had a HC-II positive test. For CIN3, HC-II was positive in all cases (100%). Assuming a similar specificity level, the relative sensitivity of the HC-II test was higher when histologically confirmed high grade lesions (CIN2+ or CIN3+) were observed. HC-II test had the best * Corresponding author at: Taiba Hospital, Mubarak Alkabeer Governorate, Sabah Alsalem Area, Block 3, Street 3, P.O. Box 66482, Bayan 43755, Kuwait. Tel.: ; Mobile: address: mmorsi72@yahoo.com (M.El-Morsi Aboul-Fotouh). 1 Address: OB/GYN Dept., Faculty of Medicine, Minia University, Minia, Egypt. Peer review under responsibility of the Middle East Fertility Society. Production and hosting by Elsevier Ó 2013 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.

2 Clinical validation of high risk HPV DNA testing versus ThinPrep cytology for primary cervical cancer screening 103 sensitivity when defining cases as CIN2+ or CIN3+ (98.7% and 100%, respectively). When using the ASCUS+ cytological cutoff, the differences in CIN2+ and CIN3+ sensitivity between HC-II test and ThinPrep cytology were statistically not significant. Specificity of the ThinPrep cytology for any low and high grade histological lesions was clearly >95% when cytological diagnosis LSIL+ cutoff was used and nearly 100% when HSIL+ cutoff was used. All these specificity estimates were high compared with HC-II test. The specificity of the ThinPrep cytology decreased with about 10% when ASCUS+ was the cutoff. At cutoff ASCUS+, specificity of HC-II was comparable or only slightly lower than with ThinPrep ASCUS+ cytology with no statistically significant differences. Conclusions: ThinPrep smears and hr-hpv DNA detection by HC-II performed very well with regard to identifying high grade lesions. HPV DNA testing is a promising new technology for cervical cancer prevention and can be used for primary screening in conjunction with cervical cytology for women aged 30 years and older. Ó 2013 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction Cancer of the cervix uteri is the second most common cancer among women worldwide, with a crude incidence rate of 15.8 per women per year. In Kuwait, it is the sixth common cancer compared to the other cancers in women of all ages with a crude incidence rate of 3.8 per women per year (1). Clinical and epidemiological studies have identified human papilloma virus (HPV) as the central risk factor for cervical cancer development (2). To date, more than 200 HPV genotypes have been identified, but the interest is focused only on genital HPV (40 genotypes) that is associated with precancerous and cancerous lesions of the cervix (3). HPV types have been grouped into low-risk and high-risk types based on the frequency of association with invasive cervical cancer. Some HPV types considered low risk, such as six and 11, cause benign condylomas, whereas a wider number of subtypes considered as high risk have been shown to be involved in cervical carcinogenesis. Among them, HPV 16 and 18 genotypes, which are frequently found in association with cervical cancer, are considered the most oncogenic types. Other HPV genotypes as 31, 33, 35, 45, 51, 52, 58 and 59, that are also considered as carcinogenic, are less frequent in cervical carcinomas (4). HPV16 is the most prevalent type worldwide. HPV18, 45, 31 and 33 are the next most prevalent types. In Asia, HPV58 and HPV52 are the next most common after HPV16 and 18 (5). HPV infection by multiple genotypes has been reported to occur in 10 20% of HPV-positive cases (6). During the last six decades, screening programs based on Papcytology have undoubtedly reduced cervical cancer morbidity and mortality (7). In spite of its success, the Paptest is a subjective method with a limited sensitivity of 50% and high susceptibility to intraindividual and interindividual variability (8). Emergence of liquid-based cytology (LBC) as ThinPrep Pap tests in clinical screening for the precursors of cervical carcinoma has revolutionized the traditional Pap test. Most clinical series (9,10) have suggested that the ThinPrep cytology is more sensitive than the traditional Pap smear for the detection of cervical squamous intraepithelial lesions (SIL). In addition, the ThinPrep Pap test has the added benefit that a residual sample, especially in problematic cases, can be used to test for HPV DNA using the hybrid capture II (HC-II) technique (11). Furthermore, ThinPrep cytology has yielded encouraging results in the triage of LSIL, ASCUS and AGUS on the basis of HPV viral typing (12). However, other studies have failed to confirm some of the claims made for the technique (13,14). HPV DNA testing for cancer-associated HPV DNA is now accepted as a viable and valid option in the diagnosis of women with equivocal cytological findings and, in recent years, there has been an increasing interest in the use of HPV DNA testing in cervical samples from asymptomatic women without cytological abnormalities (15). DNA testing for oncogenic HPV types has been proposed for primary screening, either alone or in combination with cytology; for triage of equivocal Pap smears; and for surveillance and management of patients after colposcopy (16). However, the best way to implement HPV DNA testing in cervical cancer screening has not yet been established (17). Therefore, this study was conducted to compare the validity of the high risk HPV DNA (hr-hpv DNA) testing by HC-II test to ThinPrep Pap test for primary cervical cancer screening in a cross-sectional pilot study. 2. Patients and methods 2.1. Patient enrolment The study consisted of consecutive 1923 cervical smear samples taken from the same number of apparently healthy married women attending the Gynecology outpatient department of the Taiba Hospital, Sabah Al-Salem, Kuwait, from November 2009 till June 2011 in a cross sectional pilot study. The patients included women between the age of 30 and 70 years seeking screening for cervical cancer or attending for various gynecological disorders and offered screening Ethical issues The study was carried out after being approved by the Local Ethics Committee for Research. All women were informed of the study and a detailed written informed consent was taken from all participants prior to the study Exclusion criteria Virgins, women older than 70 and younger than 30 years, pregnant women, and women with a history of hysterectomy or lacked a cervix were excluded from the study. Women who were

3 104 M.E.-M. Aboul-Fotouh, I.T. Hana currently being followed up for a cervical lesion, had a history of cervical cancer, had undergone Pap testing during the previous year, or refused to give consent were also excluded Study design Consecutive cervical smear samples were taken for ThinPrep cytological screening and HPV DNA testing. Women who had positive results on cytological screening and a group of women with negative screening but with abnormal looking cervix underwent colposcopy and directed biopsies, and those with cervical precancerous lesions or cancer received appropriate treatment Cytological screening Pap smears were obtained by firmly rotating a cervix brush (Unimar, Wilton, CT) five times (1800 ) clockwise in the ectoand endocervical area. After the smear was made, the Cervix brush was swirled and pressed into 20 ml of methanol-based PreservCyt solution (Cytyc Corporation, Boxborough, MA) and then discarded. The solution was kept at ambient temperature for the preparation of liquid-based cervical slides (Thin- Preps; Cytyc Corporation).(18) The vials were transported to the laboratory within 30 min. The vials containing the cell suspension were processed by the ThinPrep 2000 processor (Cytyc). In the processor, cells were mildly dispersed, then collected on a polycarbonate filter (40 50 l0 3 cells) and transferred onto a glass slide within a 20-mm-diameter circle (19). The PreservCyt solution was centrifuged at 600 g for 10 min, and the cell pellet was resuspended in 30 ml solution containing a 9:1 ratio of Cytolyt (Cytyc) and glacial acetic acid and centrifuged at 600 g for 10 min. The cell pellet was again resuspended in 20 ml of methanol-based PreservCyt solution and processed in the ThinPrep processor (20). One slide per patient was prepared in the processor. ThinPrep smears were stained by the Papanicolaou technique (21) and cover slipped Interpretation of cytological results The slides were screened and interpreted by cytopathologist and classified according to the 2001 Bethesda System (22) for reporting, which included the following: normal (including reactive changes), ASCUS (atypical squamous cells of undetermined significance), ASC-H (atypical squamous cells, cannot exclude high grade squamous intraepithelial lesion), LSIL (low grade squamous intraepithelial lesion), and HSIL (high grade squamous iintraepithelial lesion), or carcinoma. All the screening and diagnosing of the smears were done without the data of HPV results HPV DNA screening For HPV DNA testing, additional cervical cells were collected with the cervical sampler brush included in the HC-II test kit. The brush was placed into a tube containing HC-II transport medium, and the tube was delivered to the screening laboratory. The transport medium containing the screening samples, that is, cervical cells including the possible HPV DNA, was processed with the supplies and reagents of the HC-II assay. Probe cocktail B (Qiagen Inc., Gaithersburg, MD), which targets 13 high-risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), was used according to the manufacturer s instructions. Results of the HPV DNA detection assay were expressed as a ratio of relative light units (rlu ratio) to the average of three positive controls, with 1.00 (equivalent to HPV DNA concentration of 1 pg/ml) as the cutoff for test positivity (i.e., a rlu ratio P1.00 was considered HPV DNA positive, and a rlu ratio <1.00 was considered HPV DNA negative). The test cutoff and internal quality assurance procedures were according to the manufacturer s instructions Colposcopy referral and management Colposcopy was performed and biopsies were taken when clinically or cytopathologically indicated. All women with cervical cytology abnormalities, atypical or higher, corresponding to ASCUS or higher on the Bethesda System, were referred to colposcopy for additional diagnostic procedures, regardless of hr-hpv DNA testing results. A group of controls with negative screening examinations but with suspicious cervix were referred to colposcopy as well. A total of 426 women were referred for colposcopies. Colposcopies were conducted according to routine procedure in the hospital, where the biopsy specimens were also analyzed. Distributions of the test results, as well as histologically confirmed findings, were drawn into the main categories (HSIL, LSIL, ASCUS, normal). Histology was graded using three-tiered designation for CIN, in which CIN1 is mild dysplasia, CIN2 is moderate dysplasia, and CIN3 is severe dysplasia and carcinoma in situ (including adenocarcinoma in situ). During the study period, all CIN1+ cervical lesions detected within the screening program were treated, usually with cryocautery, electrocautery, loop electrical excision procedure (LEEP), or cervical conization or hysterectomy as appropriate. All patients with abnormal smear and benign histopathology were followed-up cytologically after 6 12 months Statistical analysis Statistical analysis was done using Statistical Package for Social Sciences software (for Windows 17.0, SPSS Inc. Chicago, IL, USA). Data were presented as numbers and percent for parametric variables and as mean ± SD for non-parametric variables. The consistency of cytological results in these groups, obtained with the two different methods, was compared to each other using simple and weighted n coefficients. The relative test sensitivity and specificity were also calculated, using the histologically confirmed outcome as the gold standard. Ninety-five percent confidence intervals were estimated for sensitivity and specificity with a binomial test. It was also tested with a pairwise McNemar s test, whether there were differences in the sensitivity estimates of the tests. The pairwise testing between the conventional and ThinPrep cytology and HPV DNA test was performed with various cutoff values in histology, but using ASCUS as the cutoff in cytology. 3. Results Complete data were available for consecutive 1923 cervical smear samples taken from eligible women. The patients characteristics of the study population are summarized in Table 1. The mean age of women was 36.6 ± 8.1 with extreme ages at

4 Clinical validation of high risk HPV DNA testing versus ThinPrep cytology for primary cervical cancer screening 105 Table 1 Patients characteristics. Patients characteristics Descriptive statistics Age: Range Mean ± SD 36.6 ± 8.1 Residence: n (%) Urban 1820 (94.6) Rural 103 (5.4) Education: n (%) Illiterate 580 (30.2) Educated 1343 (69.8) Occupation: n (%) House wife 1220 (63.4) Working women 703 (36.6) Parity: n (%) <3 children 850 (44.2) P3 children 1073 (55.8) Use of contraceptive methods: n (%) Pills 550 (28.6) IUD 395 (20.5) Sexual partner: n (%) Single 1910 (99.3) Multiple 13 (0.7) +ve history of abortion: n(%) 625 (32.5) Table 2 Cytological diagnoses of the ThinPrep cytology, values are given as n (%). Cytological diagnosis * ThinPrep (%) Benign or normal 1584 (82.3) ASCUS 153 (7.9) ASC-H 49 (2.5) LSIL (including HPV/mild dysplasia/cin1) 98 (5) HSIL (including moderate and severe 16 (0.8) dysplasia, CIS, CIN2 and CIN3) Carcinoma 0 (0) Not detectable 23 (1.2) Total 1923 (100) * Bethesda system 2001 for classification of Pap smear. 30 and 70 years. More than 90% lived in an urban area. All of them were married, housewife, considerably educated and had been pregnant. The majority of the participant women reported one lifetime sexual partner. A history of contraceptive use was reported by 49.1% of women, including 20.5% using an intrauterine device and 28.6% using an oral contraceptive. A history of abortion was reported by 32.5% of women. Table 2 summarizes the cytological results of the consecutive 1923 cervical smear samples taken from eligible women. 82.3% had normal cytology (1584/1923), 153 (7.9%) cases showed ASCUS, 49 (2.5%) cases were ASC-H, whereas LSIL was found in 98 (5%) cases and HSIL were found in 16 (0.8%) cases. LSIL or more severe lesions were found in 114 (5.9%) cases. In this cohort, no cytology indicating cervical cancer was observed. Hr-HPV DNA (1 rlu ratio cutoff) was found positive in 15.5% of cases (298/1923) diagnosed cytologically. Of these 298 cases, 252 (84.5%) had normal cytological screening while 46 (15.5%) had abnormal cytology. The histopathlogical data were obtained from 426 women who underwent colposcopy. Low grade lesions (HPV changes or mild dysplasia, i.e. CIN1) were found in 174/426 (40.8%) biopsies, 41/426 (9.6%) biopsies showed high grade lesions (moderate and severe dysplasia and cancer in situ, i.e. CIN2, CIN3 or CIS) and no biopsies showed any cancers. Table 3 presents the comparison of cytological, HPV DNA and histological results. 135 out of 182 (74.2%) patients with ASCUS by ThinPrep smear had CIN1 in biopsy. CIN2 or higher-grade lesions were confirmed in seven out of 182 (3.8%) ThinPrep smears with ASCUS. CIN2 or higher-grade lesions were confirmed in 16 out of 26 (61.5%) ThinPrep smears with LSIL. All (100%) cases classified as HSIL had a biopsy diagnosis of either CIN2 or CIN3. Nineteen out of 22 cases (86.4.1%) with a biopsy diagnosis of CIN2 had a HC- II positive test for high risk viruses. For CIN3, HC-II was positive in all cases (100%). Sensitivities, specificities, positive and negative predictive values for ThinPrep smears and HPV DNA testing in the histological diagnosis of CIN2+ or CIN3+ are summarized in Table 4. Assuming a similar specificity level, the relative sensitivity of the HC-II test was higher when histologically confirmed high grade and cancer lesions (CIN2+ or CIN3+) were observed. HPV DNA testing had the best sensitivity when Table 3 ThinPrep and hr-hpv DNA testing versus pathologically confirmed outcomes. Screening tests Pathologically confirmed outcomes Benign or normal CIN1 CIN2 CIN3 Carcinoma Total ThinPrep cytological screening Benign or normal ASCUS or ASC-H LSIL HSIL Carcinoma Total Hr-HPV DNA testing Negative Positive (1 + rlu ratio} Total

5 106 M.E.-M. Aboul-Fotouh, I.T. Hana Table 4 Screening method Sensitivity, specificity, positive and negative predictive values of both screening methods. No. positive ThinPrep Pap PASCUS 224/1923 (11.65) PLSIL 42/1923 (2.18) PHSIL 16/1923 (0.83) Hr-HPV DNA >1 rlu ratio 298/1923 (15.5%) Sensitivity Specificity PPV NPV PCIN2 (%) PCIN3 (%) PCIN2 (%) PCIN3 (%) PCIN2 PCIN3 (%) PCIN2 PCIN3 (%) defining cases as CIN2 + or CIN3+ (98.7% for CIN2+ and 100% for CIN3+). When using the ASCUS+ cytological cutoff, the differences in CIN2+ and CIN3+ sensitivity between HPV DNA testing and ThinPrep cytology were statistically not significant. Specificity of the cytological methods for any low and high grade histological lesions was clearly >95% when cytological diagnosis LSIL+ cutoff was used (leading to referral for colposcopy) and nearly 100% when HSIL+ cutoff was used. All these specificity estimates were high compared with HR HPV DNA test. The specificity of the ThinPrep cytology decreased with about 10% when ASCUS+ was the cutoff. At cutoff, ASCUS+ specificity of HR HC-II was comparable or only slightly lower than with the ThinPrep ASCUS+ cytology with no statistically significant differences. 4. Discussion Although important reductions in cervical cancer incidence have been seen in developed countries; thanks to cytological screening, requirements for well-trained personnel and the need for repeated visits have made cytological screening expensive and logistically difficult to implement in low-resource settings (23), where more than 80% of cervical cancer cases worldwide currently occur (24). During the last two decades, alternatives to cytological screening have been evaluated in low-resource settings (18,25). Introduction of liquid-based cytology (LBC) has reduced the problem of efficiency in processing samples, but the diagnostic validity in terms of sensitivity and specificity still shows important shortcomings (7). Infection with distinct types of HPV is the primary etiologic factor in cervical carcinogenesis. This causal relationship has been exploited for the development of molecular technologies for viral detection to overcome limitations linked to cytological cervical screening. HPV testing has been suggested for primary screening, triage of equivocal Pap smears or low-grade lesions and follow-up after treatment for cervical intraepithelial neoplasia. Determination of HPV genotype, viral load, integration status and RNA expression could further improve the effectiveness of HPV-based screening and triage strategies (26). In the present study, we compared the test performance of HC-II versus the ThinPrep liquid cytology for the detection of cervical carcinoma. ThinPrep smears performed well in our study, with sensitivity and specificity in the upper range of its published historical performance (18). This could be attributed to good laboratory infrastructure, efforts to improve smear quality, and the expertise of the cytopathologist. As with the Pap test, the optimal studies to determine the sensitivity and specificity of LBC (e.g., ThinPrep) have not been done. Hartman et al. (27) and McCrory et al. (28) have reported slightly higher sensitivity and lower specificity of LBC for detecting any degree of CIN, with modestly lower specificity. On the other hand, Coste et al. (29) showed that conventional Pap testing was slightly more sensitive and specific than LBC. The evidence is also mixed about whether liquid-based techniques improve rates of test adequacy (27,28). One advantage of liquid-based cytology is that HPV testing can be done on the same preparation; one disadvantage is that liquid-based approaches are more expensive than conventional Pap testing (30). Large randomized controlled trials (RCTs) clearly establish that for cytology-based screening, LBC does not differ from conventional cytology in sensitivity, specificity, or relative CIN detection but may yield a lower proportion of unsatisfactory slides. Cost, overall screening strategy, and other considerations may also relate to local decisions on which approach to use for conducting cytology screening (31). A number of problems with cytology have been well described. These include: the quality of sample collection affects the results, the subjective nature of slide interpretation, and the repetitive nature of the reading, which can lead to a greater number of interpretive errors. The low sensitivity of cytology has major medical, economic and medico-legal implications. High coverage remains the most important factor for successful screening programs, but once coverage is high, improving the sensitivity of the screening test becomes increasingly important (15). In this study, hr-hpv DNA was found positive in 298/1923 (15.5%) of cases diagnosed cytologically. Of these 298 cases, 252 (84.5%) had normal cytological screening while 46 (15.5%) had abnormal cytology. This prevalence rate is in concordance with the overall world distribution of HPV in asymptomatic women (32,33). Unlike the studies that have observed that the increase in HPV prevalence is related to the increasing grade of squamous intraepithelial lesions (11,34), our results did not confirm the higher prevalence of HPV infection in women with abnormal cytology. This difference may be due to heterogeneity related to HPV detection methods and whether PCR-based methods or HC-II were used. Even within PCR-based methods, sensitivity and specificity show wide variation. For instance, a sam-

6 Clinical validation of high risk HPV DNA testing versus ThinPrep cytology for primary cervical cancer screening 107 ple of 196 Mozambican women with normal cytological findings showed HPV prevalence as diverse as 32.1% and 76.0% by PCR with PGMY09/11 and SPF10, respectively (35). A meta-analysis has presented that HC-II is the HPV detection method with the lowest analytical sensitivities but with a validated clinical value for screening (36). On the other hand, rates of HPV detection in samples with normal cytology vary widely in the literature in women with normal cytology and range from 4.9% to 30.4% for the HC-II test (34). The high sensitivity estimates for HPV testing in the present study, particularly for the detection of CIN3+, are generally consistent with prior studies (25,37 39). Specificity of the ThinPrep cytological method for any low and high grade histological lesions was clearly >95% when cytological diagnosis LSIL+ cutoff was used and nearly 100% when HSIL+ cutoff was used. All these specificity estimates were high compared with hr-hpv DNA test. The specificity of the cytological test decreased with about 10% when ASCUS+ was the cutoff. At cutoff, ASCUS+ specificity of HC-II was comparable or only slightly lower than with Thin- Prep ASCUS+ cytology with no statistical significant differences. These results are similar to previous reports (40,41,27). This means that, when these tests are used in practice alone as a single test in mass screening for cervical cancer, cytological smears will miss some of the cancerous or precancerous cases, while HC-II test will produce a lot of false positive cases (i.e. women with HPV infection who do not have any precancerous lesion) and more referral to colposcopy (41). Numerous studies have demonstrated that, compared with cytology, HPV DNA testing is more sensitive for identifying women who have CIN2+ (range of sensitivities 84 97%) (41,27,37 40). In one randomized trial using both Pap and HPV testing in random order among women aged years, sensitivity of HPV was 95% compared with 55% for Pap cytology. The combination of HPV and cytology had 100% sensitivity and a referral rate of 7.9% (39). The lower specificity of HPV DNA testing compared with cytology is a consideration. Among women older than 30 years, cytology had a specificity of 97% compared with 94% for HPV testing (39). The specificity of HPV DNA testing would likely be even lower among women younger than 30 years, who have more transient HPV infection that is of little consequence. Thus, detecting such women would potentially increase the number of follow-up diagnostic workups (e.g. colposcopy referral). Potential approaches to minimize over-referral with HPV DNA testing and improve specificity include: (1) triage HPV-positive results with cytology (40) or another more specific molecular assay (42); and (2) trigger further workup only after two sequential positive HPV test results because it is the persistence of carcinogenic HPV that confers the greatest risk of CIN2 3 (43). The advantages of HPV DNA testing as a screening test compared with cytological evaluation of the cervix are as follows: (i) Its higher sensitivity is particularly important in settings where women will be screened only once or twice in their lifetimes. (ii) HPV DNA testing not only identifies women with cervical disease but also those at risk for developing CIN within the next 3 10 years. This is particularly important for developing countries that might not have sufficient resources to screen all women at 5- to 10-year intervals, but might have the resources to screen a small subset of high-risk HPV DNA-positive women at more frequent intervals. (iii) The interpretation of the test is objective and does not have the inherent subjectivity of visual screening methods or cervical cytology (44). Verification bias was one of the limitations of the present study. The cross sectional sensitivity estimates, particularly of the ThinPrep cytology, are in this study likely high, because not all the women under the study were referred to colposcopy. Most studies used colposcopy or biopsy as the reference standard, neither of which is 100% sensitive for detecting preinvasive disease (45). It was not practical to get biopsy specimens from all women with negative cytology, but our strategy was to obtain biopsy specimens from a sample of women with negative cytology and suspicious cervix to calculate the likely number of cases that would have been found if all women with negative cytology had been fully investigated. Opposite to the verification bias, there is some tendency to under-estimate the relative sensitivity of the hr-hpv DNA test in the present study compared with the cytological screening methods, because there were women with negative cytology among those who are HPV positive. So, follow-up visits after the screening visit could have offered a more reliable gold standard than the cross sectional design used in this study, thus avoiding errors in the validity parameters potentially arising also from false positive or over diagnosis of the histologically confirmed precancerous lesions (41). 5. Conclusion ThinPrep cytology and HPV detection by HC-II performed very well with regard to identifying high grade lesions. HPV DNA testing is a promising new technology for cervical cancer prevention and is the most reproducible of all cervical cancer screening tests. HPV DNA testing can be used for primary screening in conjunction with cervical cytology for women aged 30 years and older. Disclosure of interests None. Contribution to authorship MEA is the study Gynecologist who collected the smears, data of the colposcopies and biopsies, and wrote the manuscript. ITH is the study cytopathologist who processed the samples and interpreted the smears and biopsies and performed the HC-II assay. Both authors conceived the idea, shared in the statistical analysis, and approved the final form of the manuscript. Acknowledgments The authors would like to thank the members of Obstetrics and Gynecology Department at Taiba Hospital for their great help in collecting the smears and samples for HPV DNA testing and performing colposcopies. Our appreciation is extended to the laboratory personel at Taiba Hospital for their effort. References (1) WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Human papillomavirus and related cancers in Kuwait. Summary, Report 2010.

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