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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv

HPV genotype prevalence in Australian women undergoing routine cervical screening by cytology status prior to implementation of an HPV vaccination program

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Sepehr N. Tabrizi a,b,e,∗,1 , Julia M.L. Brotherton a,c,d,1 , Matthew P. Stevens a,b , John R. Condon f , Peter McIntyre d , David Smith g , Suzanne M. Garland a,b,e , on behalf of the WHINURS group2 a

Regional WHO HPV Reference Laboratory, Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Parkville, Victoria, Australia Murdoch Childrens Research Institute, Parkville, Victoria, Australia c Victorian Cytology Service, Carlton, Victoria, Australia d National Centre for Immunisation Research and Surveillance, University of Sydney, Westmead, NSW, Australia e Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia f Menzies School of Health Research, Charles Darwin University, Casuarina, NT, Australia g School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, WA, Australia b

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Article history: Received 6 January 2014 Received in revised form 10 March 2014 Accepted 15 April 2014

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Keywords: Human papillomavirus Genotype Cervical cytology Cervical Cancer CIN Linear array

Background: Data on the prevalence of cervical HPV genotypes in Australia by age and by grade of cytological abnormality are sparse. Objective: Measure prevalence of HPV genotypes among 2620 Australian women by age and cytology status. Study design: Women presenting for routine Pap smear screening were recruited from diverse regions, including a significant sample of Indigenous women. DNA extracts prepared from Thinprep specimens were HPV genotyped by Roche LINEAR ARRAY HPV. Results: HPV prevalence and genotype distribution were stratified by age (mean 32.6 y) and Pap smear result (cytology normal in 86.7%). Overall HPV prevalence was 38.7% with high-risk HPV prevalence of 26.5%. Prevalence of HPV (66.3% in women < 21 y to 15.3% in women > 40 y), multiple HPV infection (45.5% in 40 y) and vaccine-targeted genotypes (HPV 6/11/16/18) (34.1% in 40 y) declined significantly with age. The six most common genotypes were: HPV 16 (8.3%), 51 (5.1%), 53 (4.7%), 62 (4.3%), 89 (3.9%) and 52 (3.8%). HR-HPV prevalence increased from 21.1% in women with normal cytology to 80.9% in those with cytologically predicted high-grade abnormalities (HGAs) (p < 0.001). The most common genotypes in women with HGAs were HPV 16 (51.1%), 18 (14.9%), 52 (12.8%), 31 (10.6%), and 33 (10.6%): all HR-types. Conclusion: Pre-vaccination cross-sectional prevalence of HR-HPV infection was high in this sample of Australian women attending for screening. HPV 16 was the commonest high-risk type detected at all ages and cytological grades. © 2014 Published by Elsevier B.V.

1. Background

Abbreviations: HPV, human papillomavirus; CIN, cervical intraepithelial neoplasia; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade intraepithelial lesion. ∗ Corresponding author at: Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Locked Bag 300, Parkville, Victoria 3052, Australia. Tel.: +61 3 8345 3672, Tel.: +61 3 9348 0796. E-mail addresses: [email protected], [email protected] (S.N. Tabrizi). 1 Joint first authors. 2 WHINURS group members are listed in ‘Acknowledgments’ section.

Carcinoma of the cervix is a significant cause of mortality in women, accounting for around 275,000 deaths worldwide annually [1]. Mortality rates have significantly reduced over time in developed countries due to successful cervical screening programs using the Papanicolaou [2] test, aimed at early detection of cytological abnormalities [3]. Infection with an oncogenic or high-risk [2] human papillomavirus (HPV) genotype is the major causative factor for development of precancerous high-grade cervical intraepithelial

http://dx.doi.org/10.1016/j.jcv.2014.04.013 1386-6532/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Tabrizi SN, et al. HPV genotype prevalence in Australian women undergoing routine cervical screening by cytology status prior to implementation of an HPV vaccination program. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.04.013

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lesions (CIN2/3) and ultimately cervical cancer [4–8]. However, the majority of cervical HPV infections are transient, asymptomatic and cleared by host immunity. There are approximately 40 known genotypes that specifically target the anogenital mucosa which are divided into low-risk (LR) and high-risk [2] types, through epidemiological association with anogenital cancers [9,10]. Virtually all cases of cervical cancer and/or high-grade CIN2/3 are preceded by persistent HR-HPV infection(s). Currently, there are two licensed prophylactic HPV vaccines; a bivalent HPV 16/18 (CervarixTM ) vaccine and a quadrivalent HPV 6/11/16/18 (Gardasil® ) one [11]. Both vaccines prevent infection with the two major oncogenic types causing 70% of cancers of the cervix, while the quadrivalent vaccine also targets HPV types causing over 90% of genital wart cases. In Australia, the quadrivalent HPV vaccine was approved for use in 2006, with free vaccination being made available for school-aged girls from April 2007 through the national HPV vaccination program (12–18 years-old) [12]. Between July 2007 and December 2009 the vaccine was also offered free to all women under the age of 27 years through community vaccination providers. The free school-based program for 12- to 13-year-old girls is ongoing, with adolescent males being added from February 2013. Prior to HPV vaccination in Australia, information on HPV typespecific prevalence across the general population was limited. Most HPV genotyping studies in Australia focused on the analysis of biopsies/liquid-based samples from patients with CIN2/3 and/or cervical cancer [13–17]. From these studies, HPV 16 and 18 were identified as the most common genotypes among women with higher grade disease, in line with international findings. However, very little is known about the prevalence of other HPV genotypes. To address, a nationwide study of HPV prevalence in sexually active Australian women presenting for Pap testing at 34 sites across the country was established: the Women Human papillomavirus Indigenous Non-indigenous Urban Rural Study (WHINURS) [18]. The aim of WHINURS was to ascertain HPV genotype prevalence rates among Australian women presenting for routine Pap smears across Australia, prior to rollout of the HPV prophylactic vaccines. Population based estimates adjusted for risk factors for women up to age 40 have been presented elsewhere and address the primary research questions regarding possible differences between Indigenous and remote dwelling women compared to other Australian women [18].

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2. Objectives

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To provide more comprehensive data on the age-related distribution of HPV genotypes in Australian women prior to vaccination by including women up to age 60, and to determine the association between genotypes and cytological changes on Pap smears. 3. Study design

for the study from the relevant ethics committee at the 34 sites and the Research and Ethics Committees at the Royal Women’s Hospital, Melbourne. 3.2. HPV testing DNA was prepared from PreservCyt-stored specimens by MagNA Pure LC extraction [19,20] and tested as described previously [18]. Briefly, 10 ␮l of DNA was screened by HPV AMPLICOR (AMP) for the detection of HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68); with this result used for clinical reporting. All extracts negative for HR-HPV by AMP were also analyzed by an “in-house” PGMY09/11-based HPV consensus PCR/ELISA (20 ␮l of DNA) detecting mucosal HPV types, as previously described [21] Ultimately, DNA testing positive by AMP or the in-house PCR were genotyped (50 ␮l of DNA) using the HPV Linear Array® Genotyping Test (Roche) according to the manufacturer’s instructions, with minor modifications as previously reported [19,22,23]. We considered types 26, 53, 66, 73 and 82 as possible high-risk types and other types were designated low risk. 3.3. Statistical analysis Cytology results were grouped into normal cytology, low-grade abnormalities (LGA) (possible or definite low grade squamous intraepithelial abnormality) and high-grade abnormalities (HGA) (possible or definite high-grade squamous intraepithelial abnormality). No endocervical abnormalities were detected on Pap testing in this cohort of women. Statistical analyses were performed in SPSS version 22.0, with two-sided p-values calculated using either the Fisher’s exact test or chi squared as appropriate. Tests for trend used the linear by linear association measure in SPSS. P values in the summary tables are presented in three groups as p < 0.001; p > 0.001 < 0.01; and p > 0.01 25 y (n = 102)

Women ≤ 25 y (n = 21)

Women > 25 y (n = 26)

* 27 (4.4) 6 (1.0) * 79 (12.8) * 35 (5.7) · 1 (0.2) * 29 (4.7) * 17 (2.8) 10 (1.6) * 31 (5.0) # 10 (1.6) # 28 (4.5) 7 (1.1) * 45 (7.3) * 31 (5.0) * 38 (6.2) 19 (3.1) # 18 (2.9) 15 (2.4) # 20 (3.2) ˆ (2.9) 18 # 30 (4.9) ˆ (5.5) 34 0 (0.0) * 31 (5.0) 9 (1.5) * 17 (2.8) · 1 (0.2) 10 (1.6) · 4 (0.6) 3 (0.5) * 27 (4.4) # 18 (2.9) #10 (1.6) 5 (0.8) # 28 (4.5) * 38 (6.2) 4 (0.6) * 308 (49.9) *309 (50.1) # 124 (20.1) * 185 (30.0) *216 (35.0) * 121 (19.6)

18 (1.1) 5 (0.3) 52 (3.1) 27 (1.6) 0 (0.0) 27 (1.6) 12 (0.7) 13 (0.8) 24 (1.5) 6 (0.4) 32 (1.9) 21 (1.3) 47 (2.8) 36 (2.2) 47 (2.8) 36 (2.2) 15 (0.9) 23 (1.4) 22 (1.3) 23 (1.4) 40 (2.4) 58 (3.5) 0 (0.0) 26 (1.6) 12 (0.7) 8 (0.5) 0 (0.0) 32 (1.9) 5 (0.3) 7 (0.4) 20 (1.2) 20 (1.2) 6 (0.4) 24 (1.5) 35 (2.1) 40 (2.4) 3 (0.2) · 1214 (73.4) 440 (26.6) 242 (14.6) 198 (12.0) 262 (15.8) 97 (5.9)

8 (5.8) 2 (1.5) # 42 (30.7) 15 (10.9) 0 (0.0) 14 (10.2) 12 (8.8) 5 (3.6) ˆ (16.8) 23 3 (2.2) ˆ (11.7) 16 9 (6.6) 26 (19.0) # 21 (15.3) 19 (13.9) 9 (6.6) 3 (2.2) 11 (8.0) 15 (10.9) ˆ (10.2) 14 15 (10.9) 11 (8.0) 0 (0.0) 15 (10.9) 5 (3.6) 5 (3.6) 2 (1.5) 4 (2.9) 0 (0.0) 0 (0.0) 10 (7.3) 9 (6.6) 5 (3.6) 5 (3.6) 7 (5.1) 15 (10.9) 0 (0.0) # 15 (10.9) # 122 (89.1) 25 (18.2) * 97 (70.8) # 105 (76.6) # 56 (40.9)

1 (1.0) 1 (1.0) 16 (15.7) 8 (7.8) 0 (0.0) 10 (9.8) 3 (2.9) 4 (3.9) 7 (6.9) 1 (1.0) 3 (2.9) 6 (5.9) 12 (11.8) 4 (3.9) 15 (14.7) 7 (6.9) 5 (4.9) 7 (6.9) 9 (8.8) 2 (2.0) 7 (6.9) 4 (3.9) 1 (1.0) 11 (10.8) 5 (4.9) 3 (2.9) 0 (0.0) 3 (2.9) 0 (0.0) 1 (1.0) 4 (3.9) 2 (2.0) 1 (1.0) 3 (2.9) 5 (4.9) 7 (6.9) 0 (0.0) 26 (25.5) 76 (74.5) 29 (28.4) 47 (46.1) 59 (57.8) 23 (22.5)

1 (4.8) 1 (4.8) 13 (61.9) 4 (19.0) 0 (0.0) 2 (9.5) 0 (0.0) 2 (9.5) 2 (9.5) 0 (0.0) 1 (4.8) 1 (4.8) 0 (0.0) 2 (9.5) 2 (9.5) 1 (4.8) 0 (0.0) 0 (0.0) 0 (0.0) 1 (4.8) 0 (0.0) 1 (4.8) 0 (0.0) 2 (9.5) 1 (4.8) 0 (0.0) 1 (4.8) 1 (4.8) 0 (0.0) 0 (0.0) 0 (0.0) 1 (4.8) 0 (0.0) 0 (0.0) 3 (14.3) 0 (0.0) 0 (0.0) 0 (0.0) 21 (100) 10 (47.6) 11 (52.4) 19 (90.5) ˆ (81.0) 17

1 (3.8) 0 (0.0) 11 (42.3) 3 (11.5) 0 (0.0) 3 (11.5) · 5 (19.2) 0 (0.0) 1 (3.8) 0 (0.0) 3 (11.5) 1 (3.8) 0 (0.0) 4 (15.4) 1 (3.8) 3 (11.5) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.8) 1 (3.8) 0 (0.0) 0 (0.0) 2 (7.7) 1 (3.8) 1 (3.8) 1 (3.8) 2 (7.7) 0 (0.0) 0 (0.0) 3 (11.5) 2 (7.7) 1 (3.8) 2 (7.7) 2 (7.7) 2 (7.7) 1 (3.8) 1 (3.8) 25 (96.2) 11 (42.3) 14 (53.8) 19 (73.1) 12 (46.2)

ˆ (4.1) 25

36 (2.2)

7 (5.1)

5 (4.9)

# 8 (38.1)

1 (3.8)

Sixty-three samples had unreported cytology findings. High risk defined as positive for 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68. Statistically significant within cytology category between the two age groups (P < 0.05): * 0.001, ˆ 0.01,