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Characterisation of human papillomavirus (HPV) genotypes in the Azorean population, Terceira island

Characterisation of human papillomavirus (HPV) genotypes in the Azorean population, Terceira island Background: Human papillomavirus detection is very important for the evaluation of prevention strategies in cervical cancer. In the Azorean population, the virus prevalence has never been studied, and there is no data available to preview a successful outcome with HPV vaccination. In this article, our objective is to characterise the HPV genotypes in Terceira Island, contributing for the epidemiological knowledge on the virus infection. Results: Cervical samples were collected from 289 women aged 16–81 in the Gynaecological Outpatient Clinic of the Hospital de Santo Espírito de Angra do Heroísmo (HSEAH). HPV DNA was amplified by Polymerase Chain Reaction using the general consensus primers PGMYO9/ PGMY11. Commercially available Papillomavirus Clinical Arrays kits (Genomica) were used to perform HPV genotyping. 30 women were HPV positive, with a median age of 41 years old. Our results show that the overall HPV prevalence was 10.49%. Seventeen genotypes were identified, including 58.82% high risk, 17.65% low risk and 23.53% undetermined risk. Conclusion: Unlike other epidemiological studies, HPV31 was the most frequent type (26.67%) in Terceira Island, followed by HPV16 (10.00%), HPV51, HPV53, HPV70 and HPV82 (6.67%). Further studies are needed to investigate if the HPV types found in our population are associated with the risk of progression to high-grade squamous intraepithelial lesions or cervical cancer. Background To date, more than one hundred genotypes have been Human Papillomavirus (HPV) is the main responsible of identified and 40 types are sexually transmitted and infect one of the most common sexually transmitted diseases the cervix [1,2]. Most cervical infections are transient and worldwide and persistent infection is the major risk factor cause either no detectable or mild pathological changes, for the development of cervical cancer. but in some instances, infections persist and can progress Page 1 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 over the course of several years to cervical intraepithelial (HSEAH). The Ethics Committee of Hospital de Santo neoplasia (CIN), and then possibly to invasive cervical Espírito de Angra do Heroísmo, approved the study pro- cancer. tocol. Detailed epidemiological studies of HPV infection, CIN Sample collection and progression to cancer have tailored which are the Sample material was rinsed into a liquid medium (Pre- most frequent types playing a key role in cervical carcino- servCyt, Cytyc Corporation), transported to Serviço Espe- genesis (high risk types), and optimal strategies are been cializado de Epidemiologia e Biologia Molecular designed to prevent, via HPV screening and vaccination, (SEEBMO) of HSEAH and stored at 4°C before DNA the approximately 200,000 deaths caused by this disease extraction. annually worldwide [1]. DNA extraction and purification Epidemiological studies on the prevalence of HPV types in The DNA from cervical samples was extracted and puri- cervical cancer show that 50% of all cases are related to fied, using Papillomavirus Clinical Arrays (Genomica) HPV 16. The others are associated mainly with HPVs 18, kit, according to the manufacturer's instructions. Briefly, 45, 31, 33, 52, 58 and 35, including infections with both after 1 ml cell suspension centrifugation (10 minutes, 12 single and multiple types [3]. 000 rpm, room temperature), the lysis buffer and the pro- teinase K (20 mg/ml) were added on the pelleted cells and In the European Union, there were over two million inci- incubated 2–3 hours ate 56°C in a Thermomixer Comfort dent cases of cancer in 2006 and over one million cancer Eppendorf. After 10 minutes proteinase K inactivation deaths. In women, breast cancer was the most common period at 70°C, DNA purification was performed, using form of cancer, followed by colorectal cancer and there DNA purification columns and the pelleted DNA was were 82 500 (8.0%) cases of cervical cancer [4]. Portugal resuspended in 100 μl Elution Buffer. has a relatively high incidence of cervical cancer in the European Union, and the high frequency of HPV-16 infec- Polymerase chain reaction (PCR) tion is in concordance with other white populations [5]. The amplification reaction was carried out in a total vol- ume of 50 μl, containing 5 μl of extracted DNA and 45 μl Cervical cancer is largely preventable through cytological of a PCR mix with PGMYO9/PGMY11 generic consensus screening programs designed to facilitate the detection primers. The PCR mix contains, beside the generic consen- and treatment of immediate precancerous lesions. These sus primers, two internal controls that will validate each evaluations require trained cytotechnologists and up to sample genotyping: genomic DNA control and amplifica- three visits for screening [6]. Alternative methods such as tion control. The first control consists in a primer set that DNA testing for human papillomavirus, especially high- amplifies a 892 bp CFTR gene fragment and the second risk types, are becoming increasingly attractive as a pri- control amplifies 1202 bp of a modified plasmid, using mary screening tool, because of sensitivity and cost-effec- the same primers as the CFTR gene. tiveness [7]. The mixture was submitted to 45 cycles of amplification, Despite the fact that prevalent HPV types are almost the using a DNA thermalcycler T-Gradient (Biometra). Each same in all regions of the world, it is important to improve cycle included a denaturation step at 94°C for 30 seconds, HPV genotype characterisation worldwide, including followed by an annealing step at 55°C for 1 minute and a under-reported regions such as the population of the chain elongation step at 72°C for 90 seconds. Each PCR Azores, helping to define preventive strategies better. was initiated with a 9 minutes denaturation step at 95°C and finished by an 8 minutes extension-step at 72°C. The aim of this study is to participate in the global effort to characterise HPV genotypes, contributing for the fulfil- Different laboratory areas were used for sample handling ment on the epidemiology of HPV infection. (pre-PCR area), product amplification (PCR area) and HPV genotyping (post-PCR area). Methods Study population HPV testing A total of 289 women with a median age of 41 years The commercially available Papillomavirus Clinical (range 16 to 81 years) participated in this study between Arrays (Genomica) kit was used for HPV DNA genotyp- February 2006 and January 2007. The population was ing. All samples were analysed for the presence of he fol- consecutively recruited from the Gynaecology Outpatient lowing HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, Clinic at the Department of Obstetrics and Gynaecology 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, of the Hospital de Santo Espírito de Angra do Heroísmo 71, 72, 73, 81, 82, 83, 84, 85 and 89. Page 2 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 HPV detection was conducted using PGMYO9/PGMY11 A ge di s t r i bu t i o n general consensus primers designed to amplify a 450 bp HPV L1 gene fragment. This region is used because it is highly conserved between different HPV types but has suf- HPV + ficient variation for the identification of each one [2]. 20 HPV - The detection of the amplified PCR product was per- 0 15- 20- 25- 30- 35- 40- 45- 50- 55- 60- 65- 70- 75- 80- formed with a new technological platform using a low 19 24 29 34 39 44 49 54 59 64 69 74 79 84 density Microarray, anchored in 2 ml tube-AT tube. It allows simultaneous detection of multiple molecular Age-distribution Figure 1 in HPV-positive and HPV-negative women markers in the L1 fragment of 35 different HPV types and Age-distribution in HPV-positive and HPV-negative in the necessary controls to insure a feasible assay. All women. process was followed according to the manufacture's standard protocol. PCR products were marked with biotin and, after amplifi- cation, they hybridised with the respective probes in spe- Single infections were found in 26 women (86.67%) and cific known AT tube areas. Biotin binds to streptavidin- multiple infections were detected in 4 (13.33%), ranging peroxidase after incubation. The addition of the TMB sub- from 2 to 3 different genotypes (average of 1.17 types per strate (3,3',5,5'-tetrametilbinzidine) generates an insolu- women). ble product after hybridisation. The most common types of Human Papillomavirus in The results were processed by software, which allows Terceira Island were HPV31, followed by HPV16, HPV51, detection, interpretation and reporting for each sample. HPV 53, HPV70 and HPV82 (Figure 2). Results Discussion The study encompasses two hundred and eighty nine In the present study, we intended to evaluate the preva- women with a mean age of 41 years and was stratified in lence of different HPV type infection in a sample of 5-year age groups. women from the Azorean population. Among those referred to a Gynaecologic Outpatient Clinic, the preva- Two hundred and seventy five (95.2%) women had nor- lence of HPV infection in the Azorean population mal cytology. Epithelial cell abnormalities were detected (10.49%) was concordant with the range of 2 to 44% in 14 women: 4 were labelled as atypical squamous cell of world-wide [8]. undetermined significance (ASCUS; 1.38%), 7 had cervi- cal intraepithelial neoplasia (CIN; 2.42%), and 3 women Burchell et al showed that age-specific HPV prevalence (1.04%) had been treated for cervical carcinoma and were among women with normal cytology was highest for excluded from the study sample. The three exclusion cases younger women (<20) and decreased in the middle age were HPV positive: HPV16, HPV61 and 66 and HPV58. groups, to increase again at age 65 and older [9]. This find- ing is not concordant with our results were HPV preva- HPV-DNA was detected in thirty (10.49%) out of tow lence was highest between 25 and 34 years old and in hundred and eighty six eligible women and only one had middle age groups (40 to 54 years old) (Figure 1). Regard- cytological abnormalities (CIN). ing older women, there were no women on age groups 75 to 79 years old, but HPV infection decreased from 60 to 84 Figure 1 shows the age distribution of population under years old in our sample. investigation, discriminating HPV positive and negative women; two peaks in age groups 25 to 34 and in 40 to 54 The second major peak of infection identified in our pop- years old are clearly in evidence. Women younger than 25 ulation, as described in other studies, was in the peri- or years and older than 59 years had a lower HPV prevalence. post-menopausal years. Although the reason for this "menopausal peak" is still unclear, it may be related to the Seventeen HPV types were detected, encompassing three reactivation of latent infections acquired earlier in life, low-risk types (17.65%) – including a double low risk due to a gradual loss of type-specific immunity or to infection, ten high-risk types (58.82%), and four undeter- acquisition of new infections by sexual contacts with new mined risk types (23.53%). partners later in life [8,9]. Page 3 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 30,00 25,00 20,00 15,00 10,00 5,00 0,00 31 16 51 53 70 82 18 39 56 58 62 66 68 16, 31, 42, 82, 33, 66 43 83 HPV T y p e H Figure 2 uman Papilomavirus type distribution Human Papilomavirus type distribution. The most common HPV types in Clifford et al meta-anal- and non-oncogenic and can induce high levels of neutral- ysis, in either single and multiple infections, were HPV16, izing antibodies [10,11]. HPV 42, HPV 58, HPV 31, HPV 18, HPV 56, HPV 81, HPV 35, HPV 33 and HPV 45 [7]. Two vaccines are currently available and they incorporate HPV types more frequently associated with cervical can- In our sample, the majority of HPV-positive women were cer. A bivalent VLP vaccine (Cervarix™ GlaxoSmithKline) infected with the high-risk HPV 31 – 26.67% (Fig. 2), is composed of the assembled VLPs of HPV16 and HPV18 which has a distribution of 9%, 7%, 5% and 4% in L1, and a quadrivalent VLP based vaccine (Gardasil Europe, Sub-Saharan Africa, South America and Asia, Merck & Co) that includes HPV16, HPV18, HPV6 and respectively, according to the Clifford study. HPV11; these last two types cause the majority of genital warts (approximately 90%) in both men and women Rates of HPV-16 infected women in Azores (10.00%) [10]. were quite similar to those in Asia (14%) and much lower than in Europe (21%). The proportions of high-risk HPV infections preventable by a vaccine for HPV16 and HPV18 vary by region, being HPV18 infection among women was very similar across highest in Europe and lowest in Sub-Saharan Africa [12]. all world regions, varying from 4 to 5%; slightly lower However, the available vaccines do not contain HPV31, results were obtained in the Azores population (3.33%). which is prevalent in the Azorean population. The discrepancy between our data and previous European Determining whether any significant protection is pro- publications may be related to important influxes of vided between genotypes will be a prerequisite to increase groups coming from Europe; especially Eastern Europe the breadth of genotype coverage for prophylactic vac- (Ukraine and Russia), Africa (Cabo Verde), Latin America cines. If VLP vaccines are found to confer a high rate of and North America. type-specific protection but no significant cross-protec- tion, alternative second-generation vaccines may be Prevention of HPV infection through vaccination is needed to protect against other high risk HPV types, such expected to dramatically reduce the morbidity and mor- as HPV45 and HPV31, to perform a major reduction in tality associated with HPV infection. These vaccines are cervical cancer [13]. based on the major capsid protein of the virus, L1 pro- teins, which are capable of self-assembling into virus like Conclusion particles (VLPs) when expressed in cells. These VLPs share Future studies are necessary to confirm some study issues. great similarity to native HPV virions, are non-infectious It is extremely important to enlarge our sample in order to confirm the very high HPV31 prevalence not only in nor- Page 4 of 5 (page number not for citation purposes) (% ) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 L1 virus-like particle vaccine in young women: a randomised mal cytology but also in high-grade lesions and in cervical double-blind placebo-controlled multicentre phase II effi- cancer. cacy trial. Lancet Oncol 2005, 6:271-278. 12. Clifford G, Franceschi S, Diaz M, Munoz N, Villa LL: Chapter 3: HPV type-distribution in women with and without cervical neo- These findings will let us know about the efficacy of the plastic diseases. Vaccine 2006, 24:. existing vaccines and if a new one should be tailored for 13. Lowy DR, Frazer IH: Chapter 16: Prophylactic human papillo- mavirus vaccines. J Natl Cancer Inst Monogr 2003, 31:111-116. the population under investigation. Competing interests The authors declare that they have no competing interests. Authors' contributions MBA, FT and LM carried out cervical sample collection. MS, MRS and ARC carried out DNA extraction. ID carried out DNA extraction, HPV genotyping, and drafted the manuscript. SH reviewed the manuscript. JBA coordinated and reviewed manuscript. All authors read and approved the final manuscript. Acknowledgements We acknowledge the Pathology Service, from Hospital de Santo Espírito de Angra do Heroísmo. Manuscript publication was funded by DRCT – Direcção Regional de Ciência e Tecnologia (Regional Authority for Science and Technology), MANSEEBMO Project, number MI.2.1/004/2005. References 1. Herrero R, Castle PE, Schiffman M, Bratti MC, Hildesheim A, Morales J, Alfaro M, Sherman ME, Wacholder S, Chen S, Rodriguez AC, Burk RD: Epidemiologic profile of type-specific human papilloma- virus infection and cervical neoplasia in Guanacaste, Costa Rica. J Infect Dis 2005, 191:1796-1807. 2. de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H: Classification of papillomaviruses. Virology 2004, 324:17-27. 3. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003, 348:518-527. 4. Ferlay J, Autier P, Boniol M, Heanue M, Colombet M, Boyle P: Esti- mates of the cancer incidence and mortality in Europe in 2006. Ann Oncol 2007, 18:581-592. 5. Medeiros R, Prazeres H, Pinto D, Macedo-Pinto I, Lacerda M, Lopes C, Cruz E: Characterization of HPV genotype profile in squa- mous cervical lesions in Portugal, a southern European pop- ulation at high risk of cervical cancer. Eur J Cancer Prev 2005, 14:467-471. 6. Goldie SJ, Gaffikin L, Goldhaber-Fiebert JD, Gordillo-Tobar A, Levin C, Mahe C, Wright TC: Cost-effectiveness of cervical-cancer screening in five developing countries. N Engl J Med 2005, 353:2158-2168. 7. Clifford GM, Gallus S, Herrero R, Munoz N, Snijders PJ, Vaccarella S, Anh PT, Ferreccio C, Hieu NT, Matos E, Molano M, Rajkumar R, Ronco G, de Sanjose S, Shin HR, Sukvirach S, Thomas JO, Tunsakul S, Meijer CJ, Franceschi S: Worldwide distribution of human pap- Publish with Bio Med Central and every illomavirus types in cytologically normal women in the Inter- scientist can read your work free of charge national Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005, 366:991-998. "BioMed Central will be the most significant development for 8. Trottier H, Franco EL: The epidemiology of genital human pap- disseminating the results of biomedical researc h in our lifetime." illomavirus infection. Vaccine 2006, 24:1-15. Sir Paul Nurse, Cancer Research UK 9. Burchell AN, Winer RL, de Sanjose S, Franco EL: Chapter 6: Epide- miology and transmission dynamics of genital HPV infection. Your research papers will be: Vaccine 2006, 24:52-61. 10. Hymel PA: Decreasing risk: impact of HPV vaccination on out- available free of charge to the entire biomedical community comes. Am J Manag Care 2006, 12:S473-83. peer reviewed and published immediately upon acceptance 11. Villa LL, Costa RL, Petta CA, Andrade RP, Ault KA, Giuliano AR, Wheeler CM, Koutsky LA, Malm C, Lehtinen M, Skjeldestad FE, Ols- cited in PubMed and archived on PubMed Central son SE, Steinwall M, Brown DR, Kurman RJ, Ronnett BM, Stoler MH, yours — you keep the copyright Ferenczy A, Harper DM, Tamms GM, Yu J, Lupinacci L, Railkar R, Tad- deo FJ, Jansen KU, Esser MT, Sings HL, Saah AJ, Barr E: Prophylactic BioMedcentral Submit your manuscript here: quadrivalent human papillomavirus (types 6, 11, 16, and 18) http://www.biomedcentral.com/info/publishing_adv.asp Page 5 of 5 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

Characterisation of human papillomavirus (HPV) genotypes in the Azorean population, Terceira island

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Springer Journals
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Copyright © 2008 by Dutra et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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1750-9378
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10.1186/1750-9378-3-6
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18426589
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Abstract

Background: Human papillomavirus detection is very important for the evaluation of prevention strategies in cervical cancer. In the Azorean population, the virus prevalence has never been studied, and there is no data available to preview a successful outcome with HPV vaccination. In this article, our objective is to characterise the HPV genotypes in Terceira Island, contributing for the epidemiological knowledge on the virus infection. Results: Cervical samples were collected from 289 women aged 16–81 in the Gynaecological Outpatient Clinic of the Hospital de Santo Espírito de Angra do Heroísmo (HSEAH). HPV DNA was amplified by Polymerase Chain Reaction using the general consensus primers PGMYO9/ PGMY11. Commercially available Papillomavirus Clinical Arrays kits (Genomica) were used to perform HPV genotyping. 30 women were HPV positive, with a median age of 41 years old. Our results show that the overall HPV prevalence was 10.49%. Seventeen genotypes were identified, including 58.82% high risk, 17.65% low risk and 23.53% undetermined risk. Conclusion: Unlike other epidemiological studies, HPV31 was the most frequent type (26.67%) in Terceira Island, followed by HPV16 (10.00%), HPV51, HPV53, HPV70 and HPV82 (6.67%). Further studies are needed to investigate if the HPV types found in our population are associated with the risk of progression to high-grade squamous intraepithelial lesions or cervical cancer. Background To date, more than one hundred genotypes have been Human Papillomavirus (HPV) is the main responsible of identified and 40 types are sexually transmitted and infect one of the most common sexually transmitted diseases the cervix [1,2]. Most cervical infections are transient and worldwide and persistent infection is the major risk factor cause either no detectable or mild pathological changes, for the development of cervical cancer. but in some instances, infections persist and can progress Page 1 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 over the course of several years to cervical intraepithelial (HSEAH). The Ethics Committee of Hospital de Santo neoplasia (CIN), and then possibly to invasive cervical Espírito de Angra do Heroísmo, approved the study pro- cancer. tocol. Detailed epidemiological studies of HPV infection, CIN Sample collection and progression to cancer have tailored which are the Sample material was rinsed into a liquid medium (Pre- most frequent types playing a key role in cervical carcino- servCyt, Cytyc Corporation), transported to Serviço Espe- genesis (high risk types), and optimal strategies are been cializado de Epidemiologia e Biologia Molecular designed to prevent, via HPV screening and vaccination, (SEEBMO) of HSEAH and stored at 4°C before DNA the approximately 200,000 deaths caused by this disease extraction. annually worldwide [1]. DNA extraction and purification Epidemiological studies on the prevalence of HPV types in The DNA from cervical samples was extracted and puri- cervical cancer show that 50% of all cases are related to fied, using Papillomavirus Clinical Arrays (Genomica) HPV 16. The others are associated mainly with HPVs 18, kit, according to the manufacturer's instructions. Briefly, 45, 31, 33, 52, 58 and 35, including infections with both after 1 ml cell suspension centrifugation (10 minutes, 12 single and multiple types [3]. 000 rpm, room temperature), the lysis buffer and the pro- teinase K (20 mg/ml) were added on the pelleted cells and In the European Union, there were over two million inci- incubated 2–3 hours ate 56°C in a Thermomixer Comfort dent cases of cancer in 2006 and over one million cancer Eppendorf. After 10 minutes proteinase K inactivation deaths. In women, breast cancer was the most common period at 70°C, DNA purification was performed, using form of cancer, followed by colorectal cancer and there DNA purification columns and the pelleted DNA was were 82 500 (8.0%) cases of cervical cancer [4]. Portugal resuspended in 100 μl Elution Buffer. has a relatively high incidence of cervical cancer in the European Union, and the high frequency of HPV-16 infec- Polymerase chain reaction (PCR) tion is in concordance with other white populations [5]. The amplification reaction was carried out in a total vol- ume of 50 μl, containing 5 μl of extracted DNA and 45 μl Cervical cancer is largely preventable through cytological of a PCR mix with PGMYO9/PGMY11 generic consensus screening programs designed to facilitate the detection primers. The PCR mix contains, beside the generic consen- and treatment of immediate precancerous lesions. These sus primers, two internal controls that will validate each evaluations require trained cytotechnologists and up to sample genotyping: genomic DNA control and amplifica- three visits for screening [6]. Alternative methods such as tion control. The first control consists in a primer set that DNA testing for human papillomavirus, especially high- amplifies a 892 bp CFTR gene fragment and the second risk types, are becoming increasingly attractive as a pri- control amplifies 1202 bp of a modified plasmid, using mary screening tool, because of sensitivity and cost-effec- the same primers as the CFTR gene. tiveness [7]. The mixture was submitted to 45 cycles of amplification, Despite the fact that prevalent HPV types are almost the using a DNA thermalcycler T-Gradient (Biometra). Each same in all regions of the world, it is important to improve cycle included a denaturation step at 94°C for 30 seconds, HPV genotype characterisation worldwide, including followed by an annealing step at 55°C for 1 minute and a under-reported regions such as the population of the chain elongation step at 72°C for 90 seconds. Each PCR Azores, helping to define preventive strategies better. was initiated with a 9 minutes denaturation step at 95°C and finished by an 8 minutes extension-step at 72°C. The aim of this study is to participate in the global effort to characterise HPV genotypes, contributing for the fulfil- Different laboratory areas were used for sample handling ment on the epidemiology of HPV infection. (pre-PCR area), product amplification (PCR area) and HPV genotyping (post-PCR area). Methods Study population HPV testing A total of 289 women with a median age of 41 years The commercially available Papillomavirus Clinical (range 16 to 81 years) participated in this study between Arrays (Genomica) kit was used for HPV DNA genotyp- February 2006 and January 2007. The population was ing. All samples were analysed for the presence of he fol- consecutively recruited from the Gynaecology Outpatient lowing HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, Clinic at the Department of Obstetrics and Gynaecology 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, of the Hospital de Santo Espírito de Angra do Heroísmo 71, 72, 73, 81, 82, 83, 84, 85 and 89. Page 2 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 HPV detection was conducted using PGMYO9/PGMY11 A ge di s t r i bu t i o n general consensus primers designed to amplify a 450 bp HPV L1 gene fragment. This region is used because it is highly conserved between different HPV types but has suf- HPV + ficient variation for the identification of each one [2]. 20 HPV - The detection of the amplified PCR product was per- 0 15- 20- 25- 30- 35- 40- 45- 50- 55- 60- 65- 70- 75- 80- formed with a new technological platform using a low 19 24 29 34 39 44 49 54 59 64 69 74 79 84 density Microarray, anchored in 2 ml tube-AT tube. It allows simultaneous detection of multiple molecular Age-distribution Figure 1 in HPV-positive and HPV-negative women markers in the L1 fragment of 35 different HPV types and Age-distribution in HPV-positive and HPV-negative in the necessary controls to insure a feasible assay. All women. process was followed according to the manufacture's standard protocol. PCR products were marked with biotin and, after amplifi- cation, they hybridised with the respective probes in spe- Single infections were found in 26 women (86.67%) and cific known AT tube areas. Biotin binds to streptavidin- multiple infections were detected in 4 (13.33%), ranging peroxidase after incubation. The addition of the TMB sub- from 2 to 3 different genotypes (average of 1.17 types per strate (3,3',5,5'-tetrametilbinzidine) generates an insolu- women). ble product after hybridisation. The most common types of Human Papillomavirus in The results were processed by software, which allows Terceira Island were HPV31, followed by HPV16, HPV51, detection, interpretation and reporting for each sample. HPV 53, HPV70 and HPV82 (Figure 2). Results Discussion The study encompasses two hundred and eighty nine In the present study, we intended to evaluate the preva- women with a mean age of 41 years and was stratified in lence of different HPV type infection in a sample of 5-year age groups. women from the Azorean population. Among those referred to a Gynaecologic Outpatient Clinic, the preva- Two hundred and seventy five (95.2%) women had nor- lence of HPV infection in the Azorean population mal cytology. Epithelial cell abnormalities were detected (10.49%) was concordant with the range of 2 to 44% in 14 women: 4 were labelled as atypical squamous cell of world-wide [8]. undetermined significance (ASCUS; 1.38%), 7 had cervi- cal intraepithelial neoplasia (CIN; 2.42%), and 3 women Burchell et al showed that age-specific HPV prevalence (1.04%) had been treated for cervical carcinoma and were among women with normal cytology was highest for excluded from the study sample. The three exclusion cases younger women (<20) and decreased in the middle age were HPV positive: HPV16, HPV61 and 66 and HPV58. groups, to increase again at age 65 and older [9]. This find- ing is not concordant with our results were HPV preva- HPV-DNA was detected in thirty (10.49%) out of tow lence was highest between 25 and 34 years old and in hundred and eighty six eligible women and only one had middle age groups (40 to 54 years old) (Figure 1). Regard- cytological abnormalities (CIN). ing older women, there were no women on age groups 75 to 79 years old, but HPV infection decreased from 60 to 84 Figure 1 shows the age distribution of population under years old in our sample. investigation, discriminating HPV positive and negative women; two peaks in age groups 25 to 34 and in 40 to 54 The second major peak of infection identified in our pop- years old are clearly in evidence. Women younger than 25 ulation, as described in other studies, was in the peri- or years and older than 59 years had a lower HPV prevalence. post-menopausal years. Although the reason for this "menopausal peak" is still unclear, it may be related to the Seventeen HPV types were detected, encompassing three reactivation of latent infections acquired earlier in life, low-risk types (17.65%) – including a double low risk due to a gradual loss of type-specific immunity or to infection, ten high-risk types (58.82%), and four undeter- acquisition of new infections by sexual contacts with new mined risk types (23.53%). partners later in life [8,9]. Page 3 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 30,00 25,00 20,00 15,00 10,00 5,00 0,00 31 16 51 53 70 82 18 39 56 58 62 66 68 16, 31, 42, 82, 33, 66 43 83 HPV T y p e H Figure 2 uman Papilomavirus type distribution Human Papilomavirus type distribution. The most common HPV types in Clifford et al meta-anal- and non-oncogenic and can induce high levels of neutral- ysis, in either single and multiple infections, were HPV16, izing antibodies [10,11]. HPV 42, HPV 58, HPV 31, HPV 18, HPV 56, HPV 81, HPV 35, HPV 33 and HPV 45 [7]. Two vaccines are currently available and they incorporate HPV types more frequently associated with cervical can- In our sample, the majority of HPV-positive women were cer. A bivalent VLP vaccine (Cervarix™ GlaxoSmithKline) infected with the high-risk HPV 31 – 26.67% (Fig. 2), is composed of the assembled VLPs of HPV16 and HPV18 which has a distribution of 9%, 7%, 5% and 4% in L1, and a quadrivalent VLP based vaccine (Gardasil Europe, Sub-Saharan Africa, South America and Asia, Merck & Co) that includes HPV16, HPV18, HPV6 and respectively, according to the Clifford study. HPV11; these last two types cause the majority of genital warts (approximately 90%) in both men and women Rates of HPV-16 infected women in Azores (10.00%) [10]. were quite similar to those in Asia (14%) and much lower than in Europe (21%). The proportions of high-risk HPV infections preventable by a vaccine for HPV16 and HPV18 vary by region, being HPV18 infection among women was very similar across highest in Europe and lowest in Sub-Saharan Africa [12]. all world regions, varying from 4 to 5%; slightly lower However, the available vaccines do not contain HPV31, results were obtained in the Azores population (3.33%). which is prevalent in the Azorean population. The discrepancy between our data and previous European Determining whether any significant protection is pro- publications may be related to important influxes of vided between genotypes will be a prerequisite to increase groups coming from Europe; especially Eastern Europe the breadth of genotype coverage for prophylactic vac- (Ukraine and Russia), Africa (Cabo Verde), Latin America cines. If VLP vaccines are found to confer a high rate of and North America. type-specific protection but no significant cross-protec- tion, alternative second-generation vaccines may be Prevention of HPV infection through vaccination is needed to protect against other high risk HPV types, such expected to dramatically reduce the morbidity and mor- as HPV45 and HPV31, to perform a major reduction in tality associated with HPV infection. These vaccines are cervical cancer [13]. based on the major capsid protein of the virus, L1 pro- teins, which are capable of self-assembling into virus like Conclusion particles (VLPs) when expressed in cells. These VLPs share Future studies are necessary to confirm some study issues. great similarity to native HPV virions, are non-infectious It is extremely important to enlarge our sample in order to confirm the very high HPV31 prevalence not only in nor- Page 4 of 5 (page number not for citation purposes) (% ) Infectious Agents and Cancer 2008, 3:6 http://www.infectagentscancer.com/content/3/1/6 L1 virus-like particle vaccine in young women: a randomised mal cytology but also in high-grade lesions and in cervical double-blind placebo-controlled multicentre phase II effi- cancer. cacy trial. Lancet Oncol 2005, 6:271-278. 12. Clifford G, Franceschi S, Diaz M, Munoz N, Villa LL: Chapter 3: HPV type-distribution in women with and without cervical neo- These findings will let us know about the efficacy of the plastic diseases. Vaccine 2006, 24:. existing vaccines and if a new one should be tailored for 13. Lowy DR, Frazer IH: Chapter 16: Prophylactic human papillo- mavirus vaccines. J Natl Cancer Inst Monogr 2003, 31:111-116. the population under investigation. Competing interests The authors declare that they have no competing interests. Authors' contributions MBA, FT and LM carried out cervical sample collection. MS, MRS and ARC carried out DNA extraction. ID carried out DNA extraction, HPV genotyping, and drafted the manuscript. SH reviewed the manuscript. JBA coordinated and reviewed manuscript. All authors read and approved the final manuscript. Acknowledgements We acknowledge the Pathology Service, from Hospital de Santo Espírito de Angra do Heroísmo. Manuscript publication was funded by DRCT – Direcção Regional de Ciência e Tecnologia (Regional Authority for Science and Technology), MANSEEBMO Project, number MI.2.1/004/2005. References 1. Herrero R, Castle PE, Schiffman M, Bratti MC, Hildesheim A, Morales J, Alfaro M, Sherman ME, Wacholder S, Chen S, Rodriguez AC, Burk RD: Epidemiologic profile of type-specific human papilloma- virus infection and cervical neoplasia in Guanacaste, Costa Rica. J Infect Dis 2005, 191:1796-1807. 2. de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H: Classification of papillomaviruses. Virology 2004, 324:17-27. 3. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003, 348:518-527. 4. Ferlay J, Autier P, Boniol M, Heanue M, Colombet M, Boyle P: Esti- mates of the cancer incidence and mortality in Europe in 2006. Ann Oncol 2007, 18:581-592. 5. 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"BioMed Central will be the most significant development for 8. Trottier H, Franco EL: The epidemiology of genital human pap- disseminating the results of biomedical researc h in our lifetime." illomavirus infection. Vaccine 2006, 24:1-15. Sir Paul Nurse, Cancer Research UK 9. Burchell AN, Winer RL, de Sanjose S, Franco EL: Chapter 6: Epide- miology and transmission dynamics of genital HPV infection. Your research papers will be: Vaccine 2006, 24:52-61. 10. Hymel PA: Decreasing risk: impact of HPV vaccination on out- available free of charge to the entire biomedical community comes. Am J Manag Care 2006, 12:S473-83. peer reviewed and published immediately upon acceptance 11. Villa LL, Costa RL, Petta CA, Andrade RP, Ault KA, Giuliano AR, Wheeler CM, Koutsky LA, Malm C, Lehtinen M, Skjeldestad FE, Ols- cited in PubMed and archived on PubMed Central son SE, Steinwall M, Brown DR, Kurman RJ, Ronnett BM, Stoler MH, yours — you keep the copyright Ferenczy A, Harper DM, Tamms GM, Yu J, Lupinacci L, Railkar R, Tad- deo FJ, Jansen KU, Esser MT, Sings HL, Saah AJ, Barr E: Prophylactic BioMedcentral Submit your manuscript here: quadrivalent human papillomavirus (types 6, 11, 16, and 18) http://www.biomedcentral.com/info/publishing_adv.asp Page 5 of 5 (page number not for citation purposes)

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Infectious Agents and Cancer – Springer Journals

Published: Apr 21, 2008

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