Journal of Medical Virology 86:1905–1910 (2014)
HPV Prevalence and Type Distribution in Women With Normal or Abnormal Pap Smear in Bulgaria Peter Grozdanov,1 Victor Zlatkov,2 Gancho Ganchev,3 Ilko Karagiosov,4 Draga Toncheva,5 and Angel S. Galabov6* 1
Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Gynecology and Obstetrics Majchin Dom Hospital, Sofia, Bulgaria 3 National Institute of Oncology and Hematology, Sofia, Bulgaria 4 Tokuda Hospital, Sofia, Bulgaria 5 Department of Medical Genetics, Medical University, Sofia, Bulgaria 6 Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria 2
Human papillomavirus (HPV) is a well-known pathogen for lower genital tract neoplasias, yet little is known regarding HPV prevalence in Bulgaria. The aim of this study was to investigate the prevalence of HPV DNA and to determine HPV types distribution among women with normal and abnormal cytology. Cervical smears with different cytological diagnoses were collected from 355 Bulgarian patients. The cohort of patients selected is the biggest ever studied in this country. Using the Roche Linear Array HPV Genotyping Test, papillomavirus DNA was found in 217 out of the 355 samples, 164 of which had only one and 53 had more than one HPV type. The distribution of the viruses tested in 355 samples was as follows: (i) the most common type was HPV 16, which was found in 61 samples; (ii) the next most frequent HPV type was HPV 33, found in 14 of the samples. A high prevalence of HPV infection was observed in this study. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Currently, two HPV vaccines are included in the National immunization schedule in Bulgaria. Thus, new clinical studies will benefit from patient stratification by the presence or absence of HPV, and by designing separate clinical trials specifically for HPV associated cancers. J. Med. Virol. 86:1905–1910, 2014. # 2014 Wiley Periodicals, Inc. KEY WORDS:
human papillomavirus; genotyping; linear array; abnormal Pap; cervical cancer; HPV vaccine
C 2014 WILEY PERIODICALS, INC.
INTRODUCTION Cervical cancer is one of the leading causes of cancer-related mortality among women on a world scale. It is estimated that 500,000 cases occur annually and the case fatality rate is 50% [Baleriola et al., 2008]. An essential etiological factor found for cervical cancer is human papillomavirus (HPV) [Bouvard et al., 2009]. The HPV infection persists in 5– 10% of infected women and leads to a high risk of development of precancerous lesions of cervix uteri, which can progress to invasive cervical cancer. Highrisk HPV types are detected in 99% of the cases with cervical cancer. Approximately 15 anogenital types among the 200 HPV types estimated, are associated with cervical cancer and are termed high-risk HPV (HR-HPV) [Mu~ noz et al., 2003; Khan et al., 2005; O’Leary et al., 2011]. HR-HPV DNA testing has been proposed as a tool for primary cancer screening [zur Hausen, 2002; Castle et al., 2008] or as an adjunct test for the treatment of patients with the equivocal cytological finding of atypical squamous cells of undetermined significance (ASC-US) [Dursun et al., 2009]. It has been demonstrated that specific identification of HR-HPV can highlight patients at greater risk for developing cervical intraepithelial neoplasia of grade 3 or higher [Gravitt et al., 1998, 2000;
The work was performed at The Stephan Angeloff Institute of Microbiology. Correspondence to: Angel S. Galabov, The Stephan Angeloff Institute of Microbiology, 26 Georgi Bonchev, 1113 Sofia, Bulgaria. E-mail: [email protected] Accepted 3 June 2014 DOI 10.1002/jmv.24020 Published online 25 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
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Grozdanov et al.
TaeJeong et al., 2004; Todorova et al., 2006; van Hamont et al., 2006; Halfon et al., 2010]. With that end in view, HR-HPV cocktail tests such as the Linear Array (LA) test (Roche) are usually employed. This test detects the presence of HR-HPV and does distinguish the genotype present. The Linear Array genotyping test is a reverse line blot assay for detection and genotyping of 37 types of HPV [Lenselink et al., 2008; Monsonego et al., 2008; Iftner et al., 2009]. The LA test is the standard for assessment of new HPV genotyping tests [Meijer et al., 2006; Liu et al., 2009; Mariani et al., 2010; Koshiol et al., 2013; Lan et al., 2013]. The present study aimed to investigate the prevalence of HPV in Bulgarian patients with different cytological diagnoses. These data, along with the evidence from a previous study [Georgiev et al., 2003] will be used in order to help the accurate informing of the recommended age of HPV vaccination in a future National vaccination program in Bulgaria.
containing immobilized oligonucleotide probes. The results were interpreted by using the reference guide and reading the matching individual types down the length of the strip.
MATERIALS AND METHODS
Cervical smears with different cytological diagnoses were collected from 355 Bulgarian patients in the following three hospitals: (i) University Hospital of Obstetrics and Gynecology Majchin Dom, Sofia; (ii) Medical Center Tokuda, Sofia; and (iii) National Institute of Oncology and Hematology, Sofia. The cohort of patients selected is the biggest ever studied in this country. The age of the patients ranged from 18 to 60 years, with an average age of 35 years. The cervical smears were classified according to the Papanicolaou classification system by the Department of Pathological Anatomy. The cytological diagnoses of the samples were as follows: (i) Pap 1 (8 samples); (ii) Pap 2 (59 samples); (iii) Pap 3 (260 samples); (iv) Pap 4 (20 samples); and (iv) Pap 5 (8 samples). For this virological study, cervical smears were used in accordance with the ethical principles applied by the Sofia’s Medical University; Tokuda Hospital, Sofia; and National Institute of Oncology and Hematology, Sofia. All legislative requirements to protect human rights were respected.
The 355 samples were selected to encompass various cytological diagnoses, including a normal one. LA testing of all samples yielded either HPV-negative or HPV-positive result. Therefore, results of the LA assay were obtained for all 355 samples. This assay was capable of detecting different, however largely overlapping sets of HPV genotypes (Table I). Papillomavirus DNA was found in 217 out of the 355 samples tested (61% of all patients) as in 164 patients’ samples (46% of all patients and 76% out of the positive patients for viral DNA) was detected only one HPV type, and in 53 patients (15% of all patients and 24% of positive patients for viral DNA) more than one papilloma virus type was found. Tables II and III summarize the detection rates of HR-HPV as related to patients’ age and cytology. The distribution of the viruses tested in the 355 samples was as follows: (i) the most common type was HPV 16, which was found in 61 samples (23% of all the samples and 37% of the positive samples); and (ii) the next most frequent HPV type was HPV 33, found in 14 of the samples (5% of all samples and 9% of the positive ones). Data from the other viral findings is shown in Table IV.
HPV Genotyping LA Assay
DISCUSSION
DNA samples were tested for HPV by the Linear Array HPV genotyping test (Roche Diagnostics, Roche Molecular Biochemicals, Meylan, France) according to the instructions of the manufacturer. The LA test uses a biotinylated PGMY09/11 primer set to amplify a 450-bp region of the L1 gene and is capable of detecting 37 HPV genotypes, including 15 HR types. DNA was amplified by PCR in a Perkin–Elmer GeneAmp PCR system 9,700 apparatus (PE Applied Biosystems, Paris, France). The denatured PCR product was subsequently hybridized to an array strip
Trends for the most common types of viruses detected in Bulgarian patients from previous studies remain identical to known exceptions [Georgiev et al., 2003]. The most frequently detected virus type in the studied samples was HPV 16 (45.6%), followed by HPV 33 (13.8%). This data on the proportion of HPV 33 was similar to the data, obtained by similar studies in other Balkan countries as FYROM (HPV 33–7.1%), Montenegro (HPV 33–7.1%), Serbia (HPV 33–7.1%), Bosnia and Herzegovina (HPV 33–7.1%), Romania (HPV 33–7.1%), and Albania (HPV 33–
Patients and Clinical Specimens
J. Med. Virol. DOI 10.1002/jmv
Interpretation of Genotyping Results The results of the LA test were read according to the instructions of the manufacturer. The results were interpreted by using the reference guide and reading the matching individual types down the length of the strip. A result was considered valid only if at least one of the globin (low and high) signal bands was visible. The LA assay offers no separate detection for HPV 52. Instead, a signal band for HPV types 33, 35, 52, and 58, and three separate bands for HPV 33, HPV 35, and HPV 58 are present. HPV 52 positivity is established only if the HPV 33/35/52/58 signal is present and the HPV 33, HPV 35, and HPV 58 signals are absent. RESULTS
HPV Prevalence in Bulgaria
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TABLE I. HPV Genotypes Recognized by GF and LA Assays Genotyping capability Oncogenic potentiala Carcinogenic (group 1)
Probably carcinogenic (group 2A) Possibly carcinogenic (group 2B)
Not subject to classification by carcinogenicity (group 3)
Genotype
LA assay
16 18 31 33 35 39 45 51 52 56 58 59 68b 26b 53 66b 67 69 70 73 82 6
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
11 40b 42 43 44 54b 55b 61b 62 64 71b 72 81 83 84b CP6108 IS39 CP8304 (81)
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
7.1%) [Todorova et al., 2006]. These results differ from those obtained in the study of patients in countries such as Turkey and Cyprus in which the second most common type detected was HPV 58 (12.2% of the patients studied), Greece HPV 31
(12.8%), and Croatia (HPV 31–9.5%) [Castle et al., 2008]. Compared to the results in other European countries, this study approaches to countries like Italy, where HPV 33 was detected as second in distribution (9%), Denmark (29.4%), and Hungary (6.7%) [Tachezy et al., 2011; WHO/ICO, 2014]. The next most common types detected were HPV 31 and HPV 6. The percentage of positive samples for HPV 6 was 7.8% and for HPV 31–9.7%. These results were closer to countries like Montenegro (HPV 31–6.8%), FYROM (HPV 31–6.8%), Serbia (HPV 31–6.8%), Bosnia and Herzegovina (HPV 31–6.8%), and Albania (HPV 31– 6.8%). The third highest type in detection was different for the various Balkan countries like Croatia (HPV 33–6.3%), Turkey (HPV 52–9.5%), Cyprus (HPV 52–9.5%), Greece (HPV 18–10.3%), and Romania (HPV 18–4.9%) [Todorova et al., 2006], compared to results obtained by the research approach in similar studies of patients from other European countries like Norway (HPV 31–9.0%), Finland (HPV 31–10.5%), Latvia (HPV 31–10.5%), Italy (HPV 31– 6.4%), Hungary (HPV 31–5.3%), and Andorra (HPV 31–6.8%) [Castle et al., 2008; Lenselink et al., 2008; Menzo et al., 2008; Mariani et al., 2010; Bernard et al., 2013]. The common viral positivity in patients distributed in age groups was ranging between 43% (40–49 years) and 77% (30–39 years). Monoinfections were prevalent in all groups. Infections with more than one HPV type were found in about 30% of cases. The number of viral types decreased with the age of the patients. The greatest variety of viral types was observed in the first age group (18–29 years)—HPV 6, 16, 18, 31, 33, 35, 39, 52, 53, 58, 60, 61, 62, 66, 81, 82, 83; in the second age group (30–39 years) 15 HPV types were found—HPV 6, 11, 16, 26, 31, 33, 45, 51, 52, 53, 56, 68, 70, 82, GTCP6908; in the third age group (40–49 years) were found 9 HPV types—HPV 16, 18, 31, 33, 49, 52, 55, 82, GTCP6108; and the lowest number of HPV types was found in the last age group (50–55 years)—HPV 16, 33, 51, 52, 58. Considering the number of viral types that infect patients with different diagnoses, a tendency was seen in the variation of this number. Five types of viruses were detected in patients with Pap 1 diagnosis—HPV 6, 16, 31, 39, 53; thirteen types of viruses were detected in patients with Pap 2 diagnosis—HPV
TABLE II. Detection Rates of HR-HPV According to Patients’ Age Number of samples
Age Group Group Group Group
1 2 3 4
(18–29) (30–39) (40–49) (50–60)
95 95 97 68
Positive for HPV DNA 60 73 42 42
(63%) (77%) (43%) (62%)
Negative for HPV DNA 35 22 55 26
(37%) (23%) (57%) (38%)
Infection with only one HPV type 40 53 29 42
(67%) (72%) (70%) (100%)
Infection with two or more HPV types 20 (33%) 20 (38%) 20 (28%) —
J. Med. Virol. DOI 10.1002/jmv
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Grozdanov et al. TABLE III. Detection Rates of HR-HPV According to Patients’ Cytology
Cytology finding Pap Pap Pap Pap Pap
1 2 3 4 5
Number of samples 8 59 260 20 8
Positive for HPV DNA 4 28 166 13 6
Negative for HPV DNA
(50%) (48%) (64%) (63%) (75%)
4 31 94 7 2
(50%) (52%) (36%) (37%) (25%)
6, 16, 31, 33, 35, 39, 58, 60, 61, 62, 66, 81, 82; and twenty eight types of viruses were found in patients with Pap 3 diagnosis—HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 49, 51, 52, 53, 55, 56, 58, 60, 61, 62, 66, 68, 70, 81, 82, 83, GTCP 6108, GTCP 6908. The results obtained for patients with Pap 4 diagnosis showed eight types of viruses—HPV 16, 81, 31, 33, 49, 52, 55, 82. The least variety of virus types detected was seen in the last group composed of patients with clinical Pap 5—HPV 16, 49, 52, 58, GTCP 6108. Virological findings in the first and second groups of patients (Pap 1 and Pap 2) were close to the results of those obtained from similar studies of patients with normal cytology conducted in Turkey, Cyprus, Romania, and Bosnia and Herzegovina. The
TABLE IV. Prevalence of HPV Types in the 355 Samples Tested
HPV types HPV6 HPV11 HPV16 HPV18 HPV26 HPV31 HPV33 HPV35 HPV39 HPV45 HPV49 HPV51 HPV52 HPV53 HPV55 HPV56 HPV58 HPV60 HPV61 HPV62 HPV66 HPV68 HPV70 HPV81 HPV82 HPV83 HPVGTPC 6908 HPVGTCP 6108
Number of Percent of Percent of different viruses viruses HPV DNA found in found in types positive patients all patients 17 1 99 11 2 21 30 8 9 2 5 5 12 7 2 2 15 1 5 2 2 2 10 2 6 1 2 5
J. Med. Virol. DOI 10.1002/jmv
7.8 0.5 45.6 5.1 0.9 9.7 13.8 3.7 4.1 0.9 2.3 2.3 5.5 3.2 0.9 0.9 6.9 0.5 2.3 0.9 0.9 0.9 4.6 0.9 2.8 0.5 0.9 2.3
5.9 0.3 34.6 3.8 0.7 7.3 10.5 2.8 3.1 0.7 1.7 1.7 4.2 2.4 0.7 0.7 5.2 0.3 1.7 0.7 0.7 0.7 3.5 0.7 2.1 0.3 0.7 1.7
Infection with only one HPV type 3 25 122 9 4
(75%) (90%) (74%) (72%) (68%)
Infection with two or more HPV types 1 (25%) 3 (10%) 44(26%) 4 (28%) 2 (32%)
rates at which viruses were found in studies of patients with similar cytology in different countries were different. For example, in Turkey and Cyprus HPV 52 was the second most frequent with 0.8%, in Romania the second most frequent was HPV 31– 3.2%, while in Bosnia and Herzegovina—HPV 66 (0.3%) [Mu~ noz et al., 2003; Dursun et al., 2009]. The results of viral diversity in patients with normal cytology overlap with aggregated data from the World Health Organization for Europe. The percentages for individual viruses found in the European population were as follows: (i) HPV 16–2.4%; (ii) HPV 31–1.4%; (iii) HPV 52–0.9%; and (iv) HPV 39–0.7%. The third group in this study includes patients diagnosed with Pap 3 and the most common viral types found here were HPV 16 (38.5%) and HPV 33 (10.9%). These results were similar to those obtained for patients with identical diagnosis in countries such as Albania (HPV 16–21.6%, HPV 33–6.0%), Romania (HPV 16– 34.5%, HPV 33–23%), Bosnia and Herzegovina (HPV 16–21.6%, HPV 33–6.8%), Serbia (HPV 16–21.6%, HPV 33–6.0%), FYROM and Montenegro (HPV 16– 21.6%, HPV 33–6.0%) [WHO/ICO, 2014]. Obviously, the patients diagnosed with Pap 3 have a wide variety of proven viral DNA. An interesting fact was observed when examining the results of the fourth group of patients (Pap 4), in which the most common type was not HPV 16, which was in third place (15.8%), but HPV 33 (26.3%) and HPV 31 (21.1%). The results for the patients with the most severe diagnosis, Pap 5, showed prevalence of the high-risk type HPV 16 (55.6%). It is important to understand the benefits and limitations of the different HPV detection methodologies in common use for appropriate interpretation of epidemiological and clinical research on HPV and HPV-related disease [Sherman et al., 2002; Mu~ noz et al., 2003; Monsonego et al., 2008; Roberts et al., 2011]. In the multiplex HPV PCR assays, the detection of multiple open reading frames for each HPV type decreases the chances for false-negative results due to the presence of genetic variants of a particular type or due to integration of the viral DNA into the host genome. The risk of false-positive results is also decreased based on the requirement for the multiplex HPV PCR assay that at least two of the ORFs amplify and are simultaneously detected to be considered as a positive sample.
HPV Prevalence in Bulgaria
In conclusion, a high prevalence of HPV infection was observed in this study. Genotyping may prove useful in stratifying HPV-positive women according to their risk of developing high-grade dysplasia, leading to tailored management strategies. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Currently, in Bulgaria, two HPV vaccines are included in the National immunization schedule. To achieve the full benefit to the patients, however, addition of HPV genotyping to screening and management protocols should not be compromised by excessive referrals for colposcopy (with inevitable over-treatment), which can be a real danger, if poorly validated HPV tests are used [Wright, 2007]. Furthermore, with the introduction of prophylactic HPV vaccines (against HPV 16 and HPV 18), a reduction of 60–70% of abnormal Pap smears can be anticipated, and genotyping for these two HPV types should have implications in monitoring these vaccine effects as well. Thus, new clinical studies will benefit from patient stratification by the presence or absence of HPV and by designing separate clinical trials specifically for HPV associated cancers. REFERENCES Baleriola C, Millar D, Melki J, Coulston N, Altman P, Rismanto N, Rawlinson W. 2008. Comparison of a novel HPV test with the Hybrid Capture II (hcII) and a reference PCR method shows high specificity and positive predictive value for 13 high-risk human papillomavirus infections. J Clin Virol 42:22–26. Bulletin of the World Health Organization (BLT): World Health Organization web site. [ http://www.who.int/bulletin/volumes/ 84/2/]. Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V. 2009. A review of human carcinogens. B. Biological agents. Lancet Oncol 10:321–322. Castle PE, Porras C, Quint WG, Rodriguez AC, Schiffman M, Gravitt PE, Gonzalez P, Katki HA, Silva S, Freer E, Van Doorn LJ, Jimenez S, Herrero R, Hildesheim A. 2008. Comparison of two PCR based human papillomavirus genotyping methods. J Clin Microbiol 46:3437–3445. Castle PE, Stoler MH, Wright TC Jr, Sharma A, Wright TL, Behrens CM. 2011. Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: A sub analysis of the ATHENA study. Lancet Oncol 12:880–890. Dursun P, Senger S, Arslan H, Kusc¸u E, Ayhan A. 2009. Human papillomavirus (HPV) prevalence and types among Turkish women at a gynecology outpatient unit. BMC Infectious Diseases 9:191. Dursun P, Senger SS, Arslan H, Kusc¸u E, Ayhan A. 2009. HPV prevalence and types among Turkish women at a gynecology outpatient unit. Infect Dis 9:191. Erik BE, Margarita PS, Michel F, Isabelle H, Elisabeth DA, Didier G, Anne CMT. 2013. Comparing human papillomavirus prevalences in women with normal cytology or invasive cervical cancer to rank genotypes according to their oncogenic potential: A meta-analysis of observational studies. BMC Infect Dis 13:373. Georgiev D, Grosdanov P, Slavchev B, Karagyozov I, Galabov AS, Toncheva D. 2003. Frequency of human papillomavirus types in Bulgarian women with cervical cancer. Int J Gynecol Obstet 15:131–137.
1909 Gravitt PE, Peyton CL, Apple RJ, Wheeler CM. 1998. Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method. J Clin Microbiol 36:3020–3027. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutle´e F, Hildesheim A, Schiffman MH, Scott DR, Apple RJ. 2000. Improved amplification of genital human papillomaviruses. J Clin Microbiol 38:357–361. Halfon P, Benmoura D, Khiri H, Penaranda G, Blanc B, Riggio D, Sandri MT. 2010. Comparison of the clinical performance of carcinogenic HPV typing of the Linear Array and Papillocheck HPV-screening assay. J Clin Virol 47:38–42. Iftner T, Germ L, Swoyer R, Kjaer SK, Breugelmans JG, Munk C, Stubenrauch F, Antonello J, Bryan JT, Taddeo FJ. 2009. Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays. J Clin Microbiol 47:2106–2113. Khan MJ, Castle PE, Lorincz AT, Wacholder S, Sherman M, Scott DR, Rush BB, Glass AG, Schiffman M. 2005. The elevated 10year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 97:1072–1079. Koshiol J, Dunn ST, Walker JL, Zuna RE, Schiffman M, Sherman ME, Gold MA, Allen RA, Zhang R, Wang SS, Wentzensen N. 2013. Reproducibility of linear array for human papillomavirus genotyping. J Clin Microbiol 51:625–628. Lan V, Dieu B, Ha L. 2013. Prevalence of cervical infection with HPV type 16 and 18 in Vietnam: Implications for vaccine campaign. BMC Cancer 13:53. Lenselink CH, Melchers WJG, Quint WGV, Hoebers A, Hendriks J, Massuger LAG, Bekkers RLM. 2008. Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in the Netherlands. PLoS ONE 3:e3743. Liu SS, Leung RC, Chan KK, Cheung AN, Ngan HY. 2009. Evaluation of a newly developed GenoArray human papillomavirus (HPV) genotyping assay by comparison with Roche Linear Array HPV genotyping assay. J Clin Microbiol 48:756–764. Mariani L, Monfulleda N, Alemany L, Vizza E, Morandino F, Vocaturo A, Benevolo M, Quiros B, lloveras B, Klaustermeier E, Quint W, Bosch F. 2010. HPV prevalence and type-specific relative contribution in invasive cervical cancer specimens from Italy. BMC Cancer 10:259. Meijer CJ, Snijders PJ, Castle PE. 2006. Clinical utility of HPV genotyping. In: Expert consensus conference: Innovations in cervical cancer prevention. Gynecol Oncol 103:12–127. Menzo S, Ciavattini A, Bagnarelli P, Marinelli K, Sisti S, Clementi M. 2008. Molecular epidemiology and pathogenic potential of under diagnosed human papillomavirus types. BMC Microbiol 8:112. Monsonego J, Pollini G, Evrard MJ, Sednaoui P, Monfort L, Quinzat D, Dachez R, Syrja¨nen K. 2008. Linear array genotyping and hybrid capture II assay in detecting human papillomavirus genotypes in women referred for colposcopy due to abnormal Papanicolaou smear. Int J STD AIDS 19:385–392. HPV typespecific prevalence using a urine assay in unvaccinated male and female 11- to 18-year olds in Scotland. Mu~ noz N, Bosch FX, de Sanjose´ S, Herrero R, Castellsague´ X, Shah KV, Snijders PJ, Meijer CJ. 2003. 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 348:518–527. O’Leary MC, Sinka K, Robertson C, Cuschieri K, Lyman R, Lacey M, Potts A, Cubie HA, Donaghy M. 2011. HPV type-specific prevalence using a urine assay in unvaccinated male and female 11- to 18-year olds in Scotland. Br J Cancer 104:1221–1226. Roberts CC, Swoyer R, Bryan JT, Taddeo FJ. 2011. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection. J Clin Microbiol 49:1899–1906. Sherman ME, Schiffman M, Cox JT. 2002. Effects of age and human papilloma viral load on colposcopy triage: Data from the randomized Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS). J Natl Cancer Inst 94:102–107. Tachezy R, Smahelova J, Salakova M, Arbyn M, Rob L, Skapa P, Jirasek T, Hamsikova E. 2011. Human papillomavirus genotype
J. Med. Virol. DOI 10.1002/jmv
1910 distribution in Czech women and men with diseases ethiologically linked to HPV. PLoS ONE 6:e21913. TaeJeong O, ChangJin K, SukKyung W, TaeSeung K, DongJun J, MyungSoon K, Sunwoo L, HyunSill C, Sungwhan A. 2004. Development and clinical evaluation of a highly sensitive DNA microarray for detection and genotyping of human papillomaviruses. J Clin Microbiol 42:3272–3280. Todorova I, Baban A, Balabanova D, Panayotova Y, Bradley J. 2006. Providers’ constructions of the role of women in cervical cancer screening in Bulgaria and Romania. Soc Sci Med 63: 776–787.
J. Med. Virol. DOI 10.1002/jmv
Grozdanov et al. van Hamont D, van Ham MA, Bakkers JM, Massuger LF, Melchers WJ. 2006. Evaluation of the SPF10-INNO LiPA human papillomavirus (HPV) genotyping test and the Roche linear array HPV genotyping test. J Clin Microbiol 44:3122–3129. WHO/ICO. Information Centre on Human Papilloma Virus (HPV) and Cervical Cancer- http://apps.who.int/hpvcentre/statistics/dynamic/ico/SummaryReportsSelect.cfm. Wright TC Jr. 2007. Cervical cancer screening in the 21st century: Is it time to retire the PAP smear? Clin Obstet Gynecol 50:313–323. zur Hausen H. 2002. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2:342–350.
HPV Prevalence and Type Distribution in Women With Normal or Abnormal Pap Smear in Bulgaria Peter Grozdanov,1 Victor Zlatkov,2 Gancho Ganchev,3 Ilko Karagiosov,4 Draga Toncheva,5 and Angel S. Galabov6* 1
Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Gynecology and Obstetrics Majchin Dom Hospital, Sofia, Bulgaria 3 National Institute of Oncology and Hematology, Sofia, Bulgaria 4 Tokuda Hospital, Sofia, Bulgaria 5 Department of Medical Genetics, Medical University, Sofia, Bulgaria 6 Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria 2
Human papillomavirus (HPV) is a well-known pathogen for lower genital tract neoplasias, yet little is known regarding HPV prevalence in Bulgaria. The aim of this study was to investigate the prevalence of HPV DNA and to determine HPV types distribution among women with normal and abnormal cytology. Cervical smears with different cytological diagnoses were collected from 355 Bulgarian patients. The cohort of patients selected is the biggest ever studied in this country. Using the Roche Linear Array HPV Genotyping Test, papillomavirus DNA was found in 217 out of the 355 samples, 164 of which had only one and 53 had more than one HPV type. The distribution of the viruses tested in 355 samples was as follows: (i) the most common type was HPV 16, which was found in 61 samples; (ii) the next most frequent HPV type was HPV 33, found in 14 of the samples. A high prevalence of HPV infection was observed in this study. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Currently, two HPV vaccines are included in the National immunization schedule in Bulgaria. Thus, new clinical studies will benefit from patient stratification by the presence or absence of HPV, and by designing separate clinical trials specifically for HPV associated cancers. J. Med. Virol. 86:1905–1910, 2014. # 2014 Wiley Periodicals, Inc. KEY WORDS:
human papillomavirus; genotyping; linear array; abnormal Pap; cervical cancer; HPV vaccine
C 2014 WILEY PERIODICALS, INC.
INTRODUCTION Cervical cancer is one of the leading causes of cancer-related mortality among women on a world scale. It is estimated that 500,000 cases occur annually and the case fatality rate is 50% [Baleriola et al., 2008]. An essential etiological factor found for cervical cancer is human papillomavirus (HPV) [Bouvard et al., 2009]. The HPV infection persists in 5– 10% of infected women and leads to a high risk of development of precancerous lesions of cervix uteri, which can progress to invasive cervical cancer. Highrisk HPV types are detected in 99% of the cases with cervical cancer. Approximately 15 anogenital types among the 200 HPV types estimated, are associated with cervical cancer and are termed high-risk HPV (HR-HPV) [Mu~ noz et al., 2003; Khan et al., 2005; O’Leary et al., 2011]. HR-HPV DNA testing has been proposed as a tool for primary cancer screening [zur Hausen, 2002; Castle et al., 2008] or as an adjunct test for the treatment of patients with the equivocal cytological finding of atypical squamous cells of undetermined significance (ASC-US) [Dursun et al., 2009]. It has been demonstrated that specific identification of HR-HPV can highlight patients at greater risk for developing cervical intraepithelial neoplasia of grade 3 or higher [Gravitt et al., 1998, 2000;
The work was performed at The Stephan Angeloff Institute of Microbiology. Correspondence to: Angel S. Galabov, The Stephan Angeloff Institute of Microbiology, 26 Georgi Bonchev, 1113 Sofia, Bulgaria. E-mail: [email protected] Accepted 3 June 2014 DOI 10.1002/jmv.24020 Published online 25 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
1906
Grozdanov et al.
TaeJeong et al., 2004; Todorova et al., 2006; van Hamont et al., 2006; Halfon et al., 2010]. With that end in view, HR-HPV cocktail tests such as the Linear Array (LA) test (Roche) are usually employed. This test detects the presence of HR-HPV and does distinguish the genotype present. The Linear Array genotyping test is a reverse line blot assay for detection and genotyping of 37 types of HPV [Lenselink et al., 2008; Monsonego et al., 2008; Iftner et al., 2009]. The LA test is the standard for assessment of new HPV genotyping tests [Meijer et al., 2006; Liu et al., 2009; Mariani et al., 2010; Koshiol et al., 2013; Lan et al., 2013]. The present study aimed to investigate the prevalence of HPV in Bulgarian patients with different cytological diagnoses. These data, along with the evidence from a previous study [Georgiev et al., 2003] will be used in order to help the accurate informing of the recommended age of HPV vaccination in a future National vaccination program in Bulgaria.
containing immobilized oligonucleotide probes. The results were interpreted by using the reference guide and reading the matching individual types down the length of the strip.
MATERIALS AND METHODS
Cervical smears with different cytological diagnoses were collected from 355 Bulgarian patients in the following three hospitals: (i) University Hospital of Obstetrics and Gynecology Majchin Dom, Sofia; (ii) Medical Center Tokuda, Sofia; and (iii) National Institute of Oncology and Hematology, Sofia. The cohort of patients selected is the biggest ever studied in this country. The age of the patients ranged from 18 to 60 years, with an average age of 35 years. The cervical smears were classified according to the Papanicolaou classification system by the Department of Pathological Anatomy. The cytological diagnoses of the samples were as follows: (i) Pap 1 (8 samples); (ii) Pap 2 (59 samples); (iii) Pap 3 (260 samples); (iv) Pap 4 (20 samples); and (iv) Pap 5 (8 samples). For this virological study, cervical smears were used in accordance with the ethical principles applied by the Sofia’s Medical University; Tokuda Hospital, Sofia; and National Institute of Oncology and Hematology, Sofia. All legislative requirements to protect human rights were respected.
The 355 samples were selected to encompass various cytological diagnoses, including a normal one. LA testing of all samples yielded either HPV-negative or HPV-positive result. Therefore, results of the LA assay were obtained for all 355 samples. This assay was capable of detecting different, however largely overlapping sets of HPV genotypes (Table I). Papillomavirus DNA was found in 217 out of the 355 samples tested (61% of all patients) as in 164 patients’ samples (46% of all patients and 76% out of the positive patients for viral DNA) was detected only one HPV type, and in 53 patients (15% of all patients and 24% of positive patients for viral DNA) more than one papilloma virus type was found. Tables II and III summarize the detection rates of HR-HPV as related to patients’ age and cytology. The distribution of the viruses tested in the 355 samples was as follows: (i) the most common type was HPV 16, which was found in 61 samples (23% of all the samples and 37% of the positive samples); and (ii) the next most frequent HPV type was HPV 33, found in 14 of the samples (5% of all samples and 9% of the positive ones). Data from the other viral findings is shown in Table IV.
HPV Genotyping LA Assay
DISCUSSION
DNA samples were tested for HPV by the Linear Array HPV genotyping test (Roche Diagnostics, Roche Molecular Biochemicals, Meylan, France) according to the instructions of the manufacturer. The LA test uses a biotinylated PGMY09/11 primer set to amplify a 450-bp region of the L1 gene and is capable of detecting 37 HPV genotypes, including 15 HR types. DNA was amplified by PCR in a Perkin–Elmer GeneAmp PCR system 9,700 apparatus (PE Applied Biosystems, Paris, France). The denatured PCR product was subsequently hybridized to an array strip
Trends for the most common types of viruses detected in Bulgarian patients from previous studies remain identical to known exceptions [Georgiev et al., 2003]. The most frequently detected virus type in the studied samples was HPV 16 (45.6%), followed by HPV 33 (13.8%). This data on the proportion of HPV 33 was similar to the data, obtained by similar studies in other Balkan countries as FYROM (HPV 33–7.1%), Montenegro (HPV 33–7.1%), Serbia (HPV 33–7.1%), Bosnia and Herzegovina (HPV 33–7.1%), Romania (HPV 33–7.1%), and Albania (HPV 33–
Patients and Clinical Specimens
J. Med. Virol. DOI 10.1002/jmv
Interpretation of Genotyping Results The results of the LA test were read according to the instructions of the manufacturer. The results were interpreted by using the reference guide and reading the matching individual types down the length of the strip. A result was considered valid only if at least one of the globin (low and high) signal bands was visible. The LA assay offers no separate detection for HPV 52. Instead, a signal band for HPV types 33, 35, 52, and 58, and three separate bands for HPV 33, HPV 35, and HPV 58 are present. HPV 52 positivity is established only if the HPV 33/35/52/58 signal is present and the HPV 33, HPV 35, and HPV 58 signals are absent. RESULTS
HPV Prevalence in Bulgaria
1907
TABLE I. HPV Genotypes Recognized by GF and LA Assays Genotyping capability Oncogenic potentiala Carcinogenic (group 1)
Probably carcinogenic (group 2A) Possibly carcinogenic (group 2B)
Not subject to classification by carcinogenicity (group 3)
Genotype
LA assay
16 18 31 33 35 39 45 51 52 56 58 59 68b 26b 53 66b 67 69 70 73 82 6
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
11 40b 42 43 44 54b 55b 61b 62 64 71b 72 81 83 84b CP6108 IS39 CP8304 (81)
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
7.1%) [Todorova et al., 2006]. These results differ from those obtained in the study of patients in countries such as Turkey and Cyprus in which the second most common type detected was HPV 58 (12.2% of the patients studied), Greece HPV 31
(12.8%), and Croatia (HPV 31–9.5%) [Castle et al., 2008]. Compared to the results in other European countries, this study approaches to countries like Italy, where HPV 33 was detected as second in distribution (9%), Denmark (29.4%), and Hungary (6.7%) [Tachezy et al., 2011; WHO/ICO, 2014]. The next most common types detected were HPV 31 and HPV 6. The percentage of positive samples for HPV 6 was 7.8% and for HPV 31–9.7%. These results were closer to countries like Montenegro (HPV 31–6.8%), FYROM (HPV 31–6.8%), Serbia (HPV 31–6.8%), Bosnia and Herzegovina (HPV 31–6.8%), and Albania (HPV 31– 6.8%). The third highest type in detection was different for the various Balkan countries like Croatia (HPV 33–6.3%), Turkey (HPV 52–9.5%), Cyprus (HPV 52–9.5%), Greece (HPV 18–10.3%), and Romania (HPV 18–4.9%) [Todorova et al., 2006], compared to results obtained by the research approach in similar studies of patients from other European countries like Norway (HPV 31–9.0%), Finland (HPV 31–10.5%), Latvia (HPV 31–10.5%), Italy (HPV 31– 6.4%), Hungary (HPV 31–5.3%), and Andorra (HPV 31–6.8%) [Castle et al., 2008; Lenselink et al., 2008; Menzo et al., 2008; Mariani et al., 2010; Bernard et al., 2013]. The common viral positivity in patients distributed in age groups was ranging between 43% (40–49 years) and 77% (30–39 years). Monoinfections were prevalent in all groups. Infections with more than one HPV type were found in about 30% of cases. The number of viral types decreased with the age of the patients. The greatest variety of viral types was observed in the first age group (18–29 years)—HPV 6, 16, 18, 31, 33, 35, 39, 52, 53, 58, 60, 61, 62, 66, 81, 82, 83; in the second age group (30–39 years) 15 HPV types were found—HPV 6, 11, 16, 26, 31, 33, 45, 51, 52, 53, 56, 68, 70, 82, GTCP6908; in the third age group (40–49 years) were found 9 HPV types—HPV 16, 18, 31, 33, 49, 52, 55, 82, GTCP6108; and the lowest number of HPV types was found in the last age group (50–55 years)—HPV 16, 33, 51, 52, 58. Considering the number of viral types that infect patients with different diagnoses, a tendency was seen in the variation of this number. Five types of viruses were detected in patients with Pap 1 diagnosis—HPV 6, 16, 31, 39, 53; thirteen types of viruses were detected in patients with Pap 2 diagnosis—HPV
TABLE II. Detection Rates of HR-HPV According to Patients’ Age Number of samples
Age Group Group Group Group
1 2 3 4
(18–29) (30–39) (40–49) (50–60)
95 95 97 68
Positive for HPV DNA 60 73 42 42
(63%) (77%) (43%) (62%)
Negative for HPV DNA 35 22 55 26
(37%) (23%) (57%) (38%)
Infection with only one HPV type 40 53 29 42
(67%) (72%) (70%) (100%)
Infection with two or more HPV types 20 (33%) 20 (38%) 20 (28%) —
J. Med. Virol. DOI 10.1002/jmv
1908
Grozdanov et al. TABLE III. Detection Rates of HR-HPV According to Patients’ Cytology
Cytology finding Pap Pap Pap Pap Pap
1 2 3 4 5
Number of samples 8 59 260 20 8
Positive for HPV DNA 4 28 166 13 6
Negative for HPV DNA
(50%) (48%) (64%) (63%) (75%)
4 31 94 7 2
(50%) (52%) (36%) (37%) (25%)
6, 16, 31, 33, 35, 39, 58, 60, 61, 62, 66, 81, 82; and twenty eight types of viruses were found in patients with Pap 3 diagnosis—HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 49, 51, 52, 53, 55, 56, 58, 60, 61, 62, 66, 68, 70, 81, 82, 83, GTCP 6108, GTCP 6908. The results obtained for patients with Pap 4 diagnosis showed eight types of viruses—HPV 16, 81, 31, 33, 49, 52, 55, 82. The least variety of virus types detected was seen in the last group composed of patients with clinical Pap 5—HPV 16, 49, 52, 58, GTCP 6108. Virological findings in the first and second groups of patients (Pap 1 and Pap 2) were close to the results of those obtained from similar studies of patients with normal cytology conducted in Turkey, Cyprus, Romania, and Bosnia and Herzegovina. The
TABLE IV. Prevalence of HPV Types in the 355 Samples Tested
HPV types HPV6 HPV11 HPV16 HPV18 HPV26 HPV31 HPV33 HPV35 HPV39 HPV45 HPV49 HPV51 HPV52 HPV53 HPV55 HPV56 HPV58 HPV60 HPV61 HPV62 HPV66 HPV68 HPV70 HPV81 HPV82 HPV83 HPVGTPC 6908 HPVGTCP 6108
Number of Percent of Percent of different viruses viruses HPV DNA found in found in types positive patients all patients 17 1 99 11 2 21 30 8 9 2 5 5 12 7 2 2 15 1 5 2 2 2 10 2 6 1 2 5
J. Med. Virol. DOI 10.1002/jmv
7.8 0.5 45.6 5.1 0.9 9.7 13.8 3.7 4.1 0.9 2.3 2.3 5.5 3.2 0.9 0.9 6.9 0.5 2.3 0.9 0.9 0.9 4.6 0.9 2.8 0.5 0.9 2.3
5.9 0.3 34.6 3.8 0.7 7.3 10.5 2.8 3.1 0.7 1.7 1.7 4.2 2.4 0.7 0.7 5.2 0.3 1.7 0.7 0.7 0.7 3.5 0.7 2.1 0.3 0.7 1.7
Infection with only one HPV type 3 25 122 9 4
(75%) (90%) (74%) (72%) (68%)
Infection with two or more HPV types 1 (25%) 3 (10%) 44(26%) 4 (28%) 2 (32%)
rates at which viruses were found in studies of patients with similar cytology in different countries were different. For example, in Turkey and Cyprus HPV 52 was the second most frequent with 0.8%, in Romania the second most frequent was HPV 31– 3.2%, while in Bosnia and Herzegovina—HPV 66 (0.3%) [Mu~ noz et al., 2003; Dursun et al., 2009]. The results of viral diversity in patients with normal cytology overlap with aggregated data from the World Health Organization for Europe. The percentages for individual viruses found in the European population were as follows: (i) HPV 16–2.4%; (ii) HPV 31–1.4%; (iii) HPV 52–0.9%; and (iv) HPV 39–0.7%. The third group in this study includes patients diagnosed with Pap 3 and the most common viral types found here were HPV 16 (38.5%) and HPV 33 (10.9%). These results were similar to those obtained for patients with identical diagnosis in countries such as Albania (HPV 16–21.6%, HPV 33–6.0%), Romania (HPV 16– 34.5%, HPV 33–23%), Bosnia and Herzegovina (HPV 16–21.6%, HPV 33–6.8%), Serbia (HPV 16–21.6%, HPV 33–6.0%), FYROM and Montenegro (HPV 16– 21.6%, HPV 33–6.0%) [WHO/ICO, 2014]. Obviously, the patients diagnosed with Pap 3 have a wide variety of proven viral DNA. An interesting fact was observed when examining the results of the fourth group of patients (Pap 4), in which the most common type was not HPV 16, which was in third place (15.8%), but HPV 33 (26.3%) and HPV 31 (21.1%). The results for the patients with the most severe diagnosis, Pap 5, showed prevalence of the high-risk type HPV 16 (55.6%). It is important to understand the benefits and limitations of the different HPV detection methodologies in common use for appropriate interpretation of epidemiological and clinical research on HPV and HPV-related disease [Sherman et al., 2002; Mu~ noz et al., 2003; Monsonego et al., 2008; Roberts et al., 2011]. In the multiplex HPV PCR assays, the detection of multiple open reading frames for each HPV type decreases the chances for false-negative results due to the presence of genetic variants of a particular type or due to integration of the viral DNA into the host genome. The risk of false-positive results is also decreased based on the requirement for the multiplex HPV PCR assay that at least two of the ORFs amplify and are simultaneously detected to be considered as a positive sample.
HPV Prevalence in Bulgaria
In conclusion, a high prevalence of HPV infection was observed in this study. Genotyping may prove useful in stratifying HPV-positive women according to their risk of developing high-grade dysplasia, leading to tailored management strategies. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Currently, in Bulgaria, two HPV vaccines are included in the National immunization schedule. To achieve the full benefit to the patients, however, addition of HPV genotyping to screening and management protocols should not be compromised by excessive referrals for colposcopy (with inevitable over-treatment), which can be a real danger, if poorly validated HPV tests are used [Wright, 2007]. Furthermore, with the introduction of prophylactic HPV vaccines (against HPV 16 and HPV 18), a reduction of 60–70% of abnormal Pap smears can be anticipated, and genotyping for these two HPV types should have implications in monitoring these vaccine effects as well. Thus, new clinical studies will benefit from patient stratification by the presence or absence of HPV and by designing separate clinical trials specifically for HPV associated cancers. REFERENCES Baleriola C, Millar D, Melki J, Coulston N, Altman P, Rismanto N, Rawlinson W. 2008. Comparison of a novel HPV test with the Hybrid Capture II (hcII) and a reference PCR method shows high specificity and positive predictive value for 13 high-risk human papillomavirus infections. J Clin Virol 42:22–26. Bulletin of the World Health Organization (BLT): World Health Organization web site. [ http://www.who.int/bulletin/volumes/ 84/2/]. Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V. 2009. A review of human carcinogens. B. Biological agents. Lancet Oncol 10:321–322. Castle PE, Porras C, Quint WG, Rodriguez AC, Schiffman M, Gravitt PE, Gonzalez P, Katki HA, Silva S, Freer E, Van Doorn LJ, Jimenez S, Herrero R, Hildesheim A. 2008. Comparison of two PCR based human papillomavirus genotyping methods. J Clin Microbiol 46:3437–3445. Castle PE, Stoler MH, Wright TC Jr, Sharma A, Wright TL, Behrens CM. 2011. Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: A sub analysis of the ATHENA study. Lancet Oncol 12:880–890. Dursun P, Senger S, Arslan H, Kusc¸u E, Ayhan A. 2009. Human papillomavirus (HPV) prevalence and types among Turkish women at a gynecology outpatient unit. BMC Infectious Diseases 9:191. Dursun P, Senger SS, Arslan H, Kusc¸u E, Ayhan A. 2009. HPV prevalence and types among Turkish women at a gynecology outpatient unit. Infect Dis 9:191. Erik BE, Margarita PS, Michel F, Isabelle H, Elisabeth DA, Didier G, Anne CMT. 2013. Comparing human papillomavirus prevalences in women with normal cytology or invasive cervical cancer to rank genotypes according to their oncogenic potential: A meta-analysis of observational studies. BMC Infect Dis 13:373. Georgiev D, Grosdanov P, Slavchev B, Karagyozov I, Galabov AS, Toncheva D. 2003. Frequency of human papillomavirus types in Bulgarian women with cervical cancer. Int J Gynecol Obstet 15:131–137.
1909 Gravitt PE, Peyton CL, Apple RJ, Wheeler CM. 1998. Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method. J Clin Microbiol 36:3020–3027. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutle´e F, Hildesheim A, Schiffman MH, Scott DR, Apple RJ. 2000. Improved amplification of genital human papillomaviruses. J Clin Microbiol 38:357–361. Halfon P, Benmoura D, Khiri H, Penaranda G, Blanc B, Riggio D, Sandri MT. 2010. Comparison of the clinical performance of carcinogenic HPV typing of the Linear Array and Papillocheck HPV-screening assay. J Clin Virol 47:38–42. Iftner T, Germ L, Swoyer R, Kjaer SK, Breugelmans JG, Munk C, Stubenrauch F, Antonello J, Bryan JT, Taddeo FJ. 2009. Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays. J Clin Microbiol 47:2106–2113. Khan MJ, Castle PE, Lorincz AT, Wacholder S, Sherman M, Scott DR, Rush BB, Glass AG, Schiffman M. 2005. The elevated 10year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 97:1072–1079. Koshiol J, Dunn ST, Walker JL, Zuna RE, Schiffman M, Sherman ME, Gold MA, Allen RA, Zhang R, Wang SS, Wentzensen N. 2013. Reproducibility of linear array for human papillomavirus genotyping. J Clin Microbiol 51:625–628. Lan V, Dieu B, Ha L. 2013. Prevalence of cervical infection with HPV type 16 and 18 in Vietnam: Implications for vaccine campaign. BMC Cancer 13:53. Lenselink CH, Melchers WJG, Quint WGV, Hoebers A, Hendriks J, Massuger LAG, Bekkers RLM. 2008. Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in the Netherlands. PLoS ONE 3:e3743. Liu SS, Leung RC, Chan KK, Cheung AN, Ngan HY. 2009. Evaluation of a newly developed GenoArray human papillomavirus (HPV) genotyping assay by comparison with Roche Linear Array HPV genotyping assay. J Clin Microbiol 48:756–764. Mariani L, Monfulleda N, Alemany L, Vizza E, Morandino F, Vocaturo A, Benevolo M, Quiros B, lloveras B, Klaustermeier E, Quint W, Bosch F. 2010. HPV prevalence and type-specific relative contribution in invasive cervical cancer specimens from Italy. BMC Cancer 10:259. Meijer CJ, Snijders PJ, Castle PE. 2006. Clinical utility of HPV genotyping. In: Expert consensus conference: Innovations in cervical cancer prevention. Gynecol Oncol 103:12–127. Menzo S, Ciavattini A, Bagnarelli P, Marinelli K, Sisti S, Clementi M. 2008. Molecular epidemiology and pathogenic potential of under diagnosed human papillomavirus types. BMC Microbiol 8:112. Monsonego J, Pollini G, Evrard MJ, Sednaoui P, Monfort L, Quinzat D, Dachez R, Syrja¨nen K. 2008. Linear array genotyping and hybrid capture II assay in detecting human papillomavirus genotypes in women referred for colposcopy due to abnormal Papanicolaou smear. Int J STD AIDS 19:385–392. HPV typespecific prevalence using a urine assay in unvaccinated male and female 11- to 18-year olds in Scotland. Mu~ noz N, Bosch FX, de Sanjose´ S, Herrero R, Castellsague´ X, Shah KV, Snijders PJ, Meijer CJ. 2003. 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 348:518–527. O’Leary MC, Sinka K, Robertson C, Cuschieri K, Lyman R, Lacey M, Potts A, Cubie HA, Donaghy M. 2011. HPV type-specific prevalence using a urine assay in unvaccinated male and female 11- to 18-year olds in Scotland. Br J Cancer 104:1221–1226. Roberts CC, Swoyer R, Bryan JT, Taddeo FJ. 2011. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection. J Clin Microbiol 49:1899–1906. Sherman ME, Schiffman M, Cox JT. 2002. Effects of age and human papilloma viral load on colposcopy triage: Data from the randomized Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS). J Natl Cancer Inst 94:102–107. Tachezy R, Smahelova J, Salakova M, Arbyn M, Rob L, Skapa P, Jirasek T, Hamsikova E. 2011. Human papillomavirus genotype
J. Med. Virol. DOI 10.1002/jmv
1910 distribution in Czech women and men with diseases ethiologically linked to HPV. PLoS ONE 6:e21913. TaeJeong O, ChangJin K, SukKyung W, TaeSeung K, DongJun J, MyungSoon K, Sunwoo L, HyunSill C, Sungwhan A. 2004. Development and clinical evaluation of a highly sensitive DNA microarray for detection and genotyping of human papillomaviruses. J Clin Microbiol 42:3272–3280. Todorova I, Baban A, Balabanova D, Panayotova Y, Bradley J. 2006. Providers’ constructions of the role of women in cervical cancer screening in Bulgaria and Romania. Soc Sci Med 63: 776–787.
J. Med. Virol. DOI 10.1002/jmv
Grozdanov et al. van Hamont D, van Ham MA, Bakkers JM, Massuger LF, Melchers WJ. 2006. Evaluation of the SPF10-INNO LiPA human papillomavirus (HPV) genotyping test and the Roche linear array HPV genotyping test. J Clin Microbiol 44:3122–3129. WHO/ICO. Information Centre on Human Papilloma Virus (HPV) and Cervical Cancer- http://apps.who.int/hpvcentre/statistics/dynamic/ico/SummaryReportsSelect.cfm. Wright TC Jr. 2007. Cervical cancer screening in the 21st century: Is it time to retire the PAP smear? Clin Obstet Gynecol 50:313–323. zur Hausen H. 2002. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2:342–350.
