Participants and procedures
A cross-sectional survey was undertaken in 2007–2009 in 11 primary health centres providing services to 9 Indigenous communities and a number of smaller outstations in the Arnhem Land region. The characteristics of this region have been described previously
. Briefly, this is a sparsely populated area in the northern part of Australia. The majority of the population are Indigenous and living in discrete remote communities of varying population size ranging from several hundred to approximately 2,500 people. Health centres based in the communities provide primary health care and limited access to visiting specialist medical services.
Communities were identified based on our earlier work
 and represented the majority in the region with vulvar cancer cases. The study was preceded by an extensive period of consultation, which included holding community forums to raise awareness of vulvar cancer. During this time an Indigenous reference group comprising Indigenous women from the region was established to advise on all aspects of the study.
Women were eligible to participate if they met the following criteria: Aboriginal and/or Torres Strait Islander, aged between 18 and 60 years, and their usual place of residence was in a community in the Arnhem Land region.
There were four methods of recruitment: (1) approaching women due for Pap screening based on health centre recall systems; (2) holding community forums; (3) holding “women’s health weeks” which had a focus on health education and screening; and (4) approaching women who presented to the health centre when the research team were present.
All participants gave written informed consent, and the study procedures conformed to the principles of the Declaration of Helsinki. Consenting women were invited to have a Pap test, specimen collection for combined vulvar/vaginal/perianal (VVP) and separate cervical HPV detection and genotyping, swabs and collection of blood (where indicated) for assessment of other sexually transmissible infections (STIs), and clinical examination for vulvar lesions. Samples for VVP genotyping were collected by clinical staff performing a single sweep swab of the vulva, vagina and peri-anal region using an established protocol
. The VVP swab was undertaken before the Pap test. Samples for endocervical HPV genotyping were taken from the PreservCyt endocervical sample collected during Pap screening. The endocervical sample was collected prior to any other swabs of the cervix or posterior fornix, using an endocervical brush that was removed carefully to avoid vaginal contamination. The majority (75%) of specimens were collected by the same clinician (MN).
Women with clinical signs of anogenital disease were referred to a gynecologist for colposcopy and biopsy for definitive histological diagnosis and treatment. Abnormal Pap and STI test results were managed by the health centre in accordance with local guidelines. Women testing positive for high-risk (HR) HPV but with a normal Pap test result were offered a repeat Pap test and HPV genotyping at 12 months.
HPV DNA testing
For cervical and VVP specimens, cellular and viral DNA was extracted using the MagNA Pure LC (MP) isolation and purification system (Roche Molecular Systems Alameda, CA, USA) with a modified protocol
. For cervical specimens, HR-HPV was detected using the Amplicor HPV test (Roche Molecular System) targeting 165 bp of the L1 gene of the 13 HR-HPV anogenital types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). Sample adequacy was determined as the Amplicor PCR method allows for simultaneous amplification of ~265 bp region of human ß-globin.
Any samples negative for HR-HPV on the Amplicor HPV test were further assessed for the presence of HR- and low risk (LR)-HPV. Briefly, 20 uL of extracted DNA was amplified for 40 cycles using 50 rmol of each of the Ll consensus primers PGMY09/11
[11, 12]. Amplification products were hybridised with a biotin-labelled HPV Ll generic probe
 and captured on streptavidin-coated plates (Roche Biochemicals)
. The bound hybrid was detected by an anti-digoxigenin peroxidase conjugate by use of the colourimetic substrate ABTS
HPV DNA genotyping
Any sample positive for HR- or LR-HPV DNA was genotyped on the Roche Linear Array (LA) HPV genotyping test (Roche Molecular Systems). This test directs amplification of a 450 bp region of the HPV L1 gene and allows identification of 37 anogenital HPV genotypes (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108) as well as amplification of 265 bp region of the human ß-globin gene, serving as an internal control. Samples were denatured and detected using a modified method as previously described
[15, 16]. Due to possible cross-reactivity of the HPV-52 probe with types 33, 35, and 58 amplicons, samples positive for the HPV-52 probe alone were classified as HPV-52 positive, whilst those reactive with this probe and one or more of HPV-33, 35, and 58 probes were further tested using a real-time PCR assay with an HPV-52 specific hydrolysis probe
, allowing detection of HPV-52 DNA in the presence/absence of other genotypes.
At the completion of the study any cervical or VVP sample that was positive on any test had the corresponding cervical or VVP sample genotyped to ensure correct comparison of differing samples. There were 10 cervical samples and 5 VVP samples that were positive on initial HPV DNA testing, but no HPV genotype was detected on LA.
The primary outcomes were the cervical and VVP prevalence of HR-HPV, defined as detection of any one of the following HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 or 59). Secondary outcomes included the cervical and VVP prevalence of: probable HR-HPV (type 68), possible HR-HPV (types 26, 53, 66, 67, 69, 70, 73 or 82), any-HPV (presence of any of the 37 types detectable on LA), LR-HPV (types 6, 11, 40, 42, 54, 55, 61, 62, 64, 71, 72, 81, 83, 84, CP6108, IS39), multiple HR types, multiple LR types, individual type-specific HPV and vaccine preventable types (6, 11, 16, 18). All HPV prevalence figures were based on HPV as detected on LA. HPV types were classified into carcinogenic groups according to the International Agency for Research on Cancer
Analyses and sample size
Proportions and means/medians were calculated to summarize the data as appropriate. As there is no data on the prevalence of vulvar HPV infection in Australian women, we compared the prevalence of cervical HR-HPV infection in Arnhem Land women to that of other Australian women (Indigenous and non-Indigenous) based on the results of the Women's HPV prevalence Indigenous Non-Indigenous Urban Rural Study (WHINURS)
. This is a national study of cervical HPV genotype prevalence undertaken between 2005–08 by members of the research team (JRC, SNT, SMG) using the same methodology (sample collection and analysis) and the same laboratory for HPV genotyping as this study. The McNemar’s matched pairs test was used to assess whether the prevalence of VVP HPV was similar to the prevalence of cervical HPV infection, in women with both samples adequate for HPV analysis. This comparison was made to provide an indirect indication of whether VVP HPV infection is more common in Arnhem Land women than other Australian women. Chi-square analysis was undertaken to assess whether HPV prevalence varied across 10-year age bands. Where the expected cell counts were less than 5, the exact significance probability is reported. A P-value <0.05 was considered statistically significant. All analyses were conducted using Stata version 10.0 (Stata Corporation, College Station, TX, USA).
The sample size was estimated using preliminary data in 2006 from the WHINURS
. To detect a difference in the prevalence of HPV-16 and 18 combined of 14.5% for Indigenous women in Arnhem Land compared with the 9.5% found in the WHINURS, 521 women were required (alpha level 0.05, 80% power, one-sided test).
This study was approved by the Human Research Ethics Committee of the Menzies School of Health Research and the Northern Territory Department of Health and Community Services, and its Aboriginal subcommittee.