Human papillomavirus genotyping using a modified linear array detection protocol

doi:10.1016/j.jviromet.2006.02.007

Journal of Virological Methods

Volume 135, Issue 1, July 2006, Pages 124-126

https://doi.org/10.1016/j.jviromet.2006.02.007 Get rights and content

Abstract

A standardized PCR-based linear array genotyping assay has been developed (Roche Molecular Systems) allowing the concurrent typing of 37 human papillomavirus (HPV) types. A component of this assay, the PCR amplicon detection protocol, requires the use of a shaking water-bath incubator for amplicon/probe hybridization and subsequent stringent washing, which many laboratories may find cumbersome or simply not possess. In this study, the utility of a dry-air incubator in substitution of the recommended shaking water-bath for use in this assay was confirmed. DNA was extracted by AmpliLute method from 150 PreservCyt specimens collected from patients undergoing ablation treatment for histologically confirmed cervical abnormality. The DNA was amplified by PCR and amplicons detected by both standard and modified protocols, varying only by the method of incubation for DNA hybridization and stringent washing. Identical HPV-type profiles were generated in 149/150 (99.3%) of the specimens tested by both protocols, representing a near perfect level of concordance (kappa = 0.98). It was proposed that this modification would markedly simplify the detection process, reduce setup time and eliminate the potential for water spillover, thereby allowing more laboratories to adopt this method subsequent to in-house validation.

Section snippets

Acknowledgements

We would like to thank Elice Rudland (Royal Women's Hospital, Carlton) for her technical assistance and Roche Molecular Systems for the contribution of reagents.

References (15)

E.M. de Villiers et al.
Classification of papillomaviruses
Virology
(2004)
N. Munoz
Human papillomavirus and cancer: the epidemiological evidence
J. Clin. Virol.
(2000)
H. zur Hausen
Human papillomaviruses in the pathogenesis of anogenital cancer
Virology
(1991)
F.X. Bosch et al.
The causal relation between human papillomavirus and cervical cancer
J. Clin. Pathol.
(2002)
G.M. Clifford et al.
Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis
Br. J. Cancer
(2003)
K.S. Cuschieri et al.
Human papillomavirus type specific DNA and RNA persistence—implications for cervical disease progression and monitoring
J. Med. Virol.
(2004)
P.E. Gravitt et al.
Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method
J. Clin. Microbiol.
(1998)

There are more references available in the full text version of this article.

Cited by (35)

Exploring monitoring strategies for population surveillance of HPV vaccine impact using primary HPV screening
2023, Tumour Virus Research
Australia's cervical screening program transitioned from cytology to HPV-testing with genotyping for HPV16/18 in Dec’2017. We investigated whether program data could be used to monitor HPV vaccination program impact (commenced in 2007) on HPV16/18 prevalence and compared estimates with pre-vaccination benchmark prevalence. Pre-vaccination samples (2005–2008) (n = 1933; WHINURS), from 25 to 64-year-old women had been previously analysed with Linear Array (LA). Post-vaccination samples (2013-2014) (n = 2989; Compass pilot), from 25 to 64-year-old women, were analysed by cobas 4800 (cobas), and by LA for historical comparability. Age standardised pre-vaccination HPV16/18 prevalence was 4.85% (95%CI:3.81–5.89) by LA; post-vaccination estimates were 1.67% (95%CI:1.21–2.13%) by LA, 1.49% (95%CI:1.05–1.93%) by cobas, and 1.63% (95%CI:1.17–2.08%) for cobas and LA testing of non-16/18 cobas positives (cobas/LA). Age-standardised pre-vaccination oncogenic HPV prevalence was 15.70% (95%CI:13.79–17.60%) by LA; post-vaccination estimates were 9.06% (95%CI:8.02–10.09%) by LA, 8.47% (95%CI:7.47–9.47%) by cobas and cobas/LA. Standardised rate ratios between post-vs. pre-vaccination rates were significantly different for HPV16/18, non-16/18 HPV and oncogenic HPV: 0.34 (95%CI:0.23–0.50), 0.68 (95%CI:0.55–0.84) and 0.58 (95%CI:0.48–0.69), respectively. Additional strategies (LA for all cobas positives; combined cobas and LA results on all samples) had similar results. If a single method is applied consistently, it will provide important data on relative changes in HPV prevalence following vaccination.
Final analysis of a study assessing genital human papillomavirus genoprevalence in young Australian women, following eight years of a national vaccination program
2018, Vaccine
Citation Excerpt :
Detection and genotyping was performed utilising PCR-based methodology, initially amplifying for the HPV L1 gene using consensus primers, with detection of amplicons by an ELISA-based system and as previously described [22]. Genotyping of those samples that were positive was by LINEAR ARRAY ®HPV Genotyping Test (Roche Diagnostics GmbH, Mannheim Germany), for which the hybridisation and washing steps were modified by using an automated blot processor (BeeBlot, Bee Robotics Ltd., Gwynedd, United Kingdom) [22–24]. This genotype testing allows for the simultaneous detection of up to 37 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 (82v) and CP6108 (HPV 89)).
The VACCINE [Vaccine Against Cervical Cancer Impact and Effectiveness] study evaluated the prevalence of quadrivalent vaccine-targeted human papillomavirus (HPV) genotypes (HPV 6, 11, 16, 18) amongst young women of vaccine-eligible age.
Between October 2011 – June 2015, women aged 18–25 years from Victoria, Australia, were recruited through targeted advertising on the social networking website Facebook. Participants completed an online questionnaire and provided a self-collected vaginal swab for HPV DNA detection and genotyping (Linear Array HPV genotyping assay). Self-reported HPV vaccination details were verified with the National HPV Vaccination Program Register (NHVPR).
Of 1223 who agreed to participate, 916 (74.9%) completed the survey and, for 1007 (82.3%) sexually-active participants, 744 (73.9%) returned the self-collected swab, of which 737 contained detectable DNA. 184/737 (25.0%) were positive for HPV. Vaccine-targeted HPV genotypes were detected in only 13 (1.7%) women: 11 HPV 16 (six vaccinated after sexual debut, five unvaccinated) and two HPV 6. Prevalence of any of HPV 31/33/45 collectively was 2.9%, varying significantly by vaccination status (fully 2.0%, unvaccinated 6.8%; p = 0.01). Vaccination rates among the sexually-active cohort were high, with 65.6%, 71.6% and 74.2% of participants having received three, at least two or at least one dose of vaccine, respectively. Of women self-reporting HPV vaccination, the NHVPR confirmed one or more doses were received in 90%. Strong associations were observed between vaccination status, age, language spoken at home and country of birth, as well as between HPV detection and the number of male sexual partners.
Surveillance five to eight years’ post-initiation of a national HPV vaccination program demonstrated a consistent and very low prevalence of vaccine-related HPV genotypes and some evidence of cross protection against related types amongst vaccine-eligible women from Victoria, Australia.
Evaluation of p16<sup>INK4a</sup> immunostaining for the detection of high-grade changes in cervical cytology
2015, Pathology
Since its introduction in Australia in 2007, the human papillo-mavirus (HPV) vaccine has led to a markedly lower prevalence of vaccine targeted HPV genotype infections as well as HPV disease including genital warts and histologically confirmed high-grade (HG) cervical abnormalities. To increase the ability to identify abnormal cells in lower prevalence, adjunct markers can be incorporated to improve the sensitivity and specificity of cytology test. One such marker is p16^INK4a(p16), which is detectable in cells expressing the E7 oncogene encoded by high-risk HPVs (HR-HPV). In this study, the sensitivity and specificity of p16 immunostaining in detection of underlying HG lesions was evaluated in a cohort of 454 women undergoing surgical treatment for biopsy proven cervical dysplasia. Overall, p16 positive cells were detected in 321 (71%) of cytology preparations evaluated. Comparison of p16 staining on cytological preprations to histology diagnosis available on 212 patients, showed 26 (54%), 41 (78%) and 80 (90%) of cytology preparations to be p16 positive in women with CIN1, CIN2 and CIN3, respectively (p < 0.005). HPV16 and 18 were the most prevalent genotypes in HG lesions and were highly correlated with p16 staining. p16 staining provides an additional marker which can assist in better detecting underlying HG lesion in cytology smears with low disease prevalence.
Assessing genital human papillomavirus genoprevalence in young Australian women following the introduction of a national vaccination program
2015, Vaccine
Citation Excerpt :
Briefly, a generic probe for detection of the presence of any HPV sequences in the sample was employed using biotin-labelled probes able to detect all mucosal HPV types [19,20]. Samples that were HPV positive by PCR-ELISA were genotyped using LINEAR ARRAY® HPV Genotyping Test (Roche Diagnostics®), for which the hybridisation and washing steps were modified by using an automated blot processor (BeeBlot, Bee Robotics) [21–23]. Genotyping profiles were interpreted manually using the HPV reference guide provided with each kit.
Following the implementation of Australia's National HPV Vaccination Program in April 2007, this study evaluated the prevalence of vaccine-targeted human papillomavirus (HPV) genotypes (HPV 6, 11, 16, 18) amongst vaccine-eligible young women.
Between September 2011 and August 2013, women from Victoria, Australia aged 18–25 were recruited through targeted advertising on the social networking website Facebook. Participants completed an online questionnaire, and sexually active women were asked to provide a self-collected vaginal swab for HPV deoxyribonucleic acid (DNA) detection and genotyping. Samples positive for HPV were genotyped using the Linear Array HPV genotyping test (Roche Diagnostics). Self-reported HPV vaccination details were verified with the National HPV Vaccination Program Register (NHVPR).
Of 431 vaginal swabs, 24.8% were positive for HPV DNA. Vaccine-targeted HPV genotypes were detected in only seven (1.6%) samples; all HPV 16 (of the six HPV 16 positive vaccinated women, all had received the vaccine after sexual debut). There were no cases of HPV 6, 11 or 18 identified. HPV types 51, 59, 73, 84, and 89 were the most prevalent genotypes. Vaccination rates were high, with 77.3% of participants having received all three doses of the vaccine, and there was an 89.8% concordance between self-reported and registry-reported HPV vaccination status. Strong associations were observed between vaccination status, age, language spoken at home and country of birth, as well as between HPV detection and the number of male sexual partners.
Preliminary data from this study demonstrate a very low prevalence of vaccine-related HPV genotypes amongst vaccine-eligible women from Victoria, Australia. We were able to use Facebook to effectively reach and recruit young women to participate in the assessment of the impact of Australia's HPV vaccination program.
Assessment of herd immunity and cross-protection after a human papillomavirus vaccination programme in Australia: A repeat cross-sectional study
2014, The Lancet Infectious Diseases
Citation Excerpt :
All samples negative for these high-risk HPV types17 were then tested for the presence of other mucosal HPV genotypes using the L1 consensus primer set PGMY09/1118 and an ELISA method to detect amplicons.19 All samples positive for HPV using any of these methods were genotyped using the LINEAR ARRAY HPV genotyping test (Roche) with modifications.20–23 Because we recruited 202 women into our prevaccine-implementation sample who were of the relevant age, we based the size of the postvaccine-implementation sample on the ability to detect herd immunity, defined by a reduction of 10% or more in the prevalence of vaccine-targeted HPV types in unvaccinated women.
After the introduction of a quadrivalent human papillomavirus (HPV) vaccination programme in Australia in April, 2007, we measured the prevalence of vaccine-targeted and closely related HPV types with the aim of assessing direct protection, cross-protection, and herd immunity.
In this repeat cross-sectional study, we recruited women aged 18–24 years who attended Pap screening between October, 2005, and July, 2007, in three major metropolitan areas of Australia to form our prevaccine-implementation sample. For our postvaccine-implementation sample, we recruited women aged 18–24 years who attended Pap screening in the same three metropolitan areas from August, 2010, to November, 2012. We compared the crude prevalence of HPV genotypes in cervical specimens between the prevaccine and the postvaccine implementation groups, with vaccination status validated against the National HPV Vaccination Program Register. We estimated adjusted prevalence ratios using log linear regression. We estimated vaccine effectiveness both for vaccine-targeted HPV types (16, 18, 6, and 11) and non-vaccine but related HPV types (31, 33, and 45).
202 women were recruited into the prevaccine-implementation group, and 1058 were recruited into the postvaccine-implementation group. Crude prevalence of vaccine-targeted HPV genotypes was significantly lower in the postvaccine-implementation sample than in the prevaccine-implementation sample (58 [29%] of 202 vs 69 [7%] of 1058; p<0·0001). Compared with the prevaccine-implementation sample, adjusted prevalence ratios for vaccine-targeted HPV genotypes were 0·07 (95% CI 0·04–0·14; p<0·0001) in fully vaccinated women and 0·65 (0·43–0·96; p=0·03) in unvaccinated women, which suggests herd immunity. No significant declines were noted for non-vaccine-targeted HPV genotypes. However, within the postvaccine-implementation sample, adjusted vaccine effectiveness against vaccine-targeted HPV types for fully vaccinated women compared with unvaccinated women was 86% (95% CI 71–93), and was 58% (26–76) against non-vaccine-targeted but related genotypes (HPV 31, 33, and 45).
6 years after the initiation of the Australian HPV vaccination programme, we have detected a substantial fall in vaccine-targeted HPV genotypes in vaccinated women; a lower prevalence of vaccine-targeted types in unvaccinated women, suggesting herd immunity; and a possible indication of cross-protection against HPV types related to the vaccine-targeted types in vaccinated women.
Australian National Health and Medical Research Council and Cancer Council Victoria.
HPV genotype prevalence in Australian women undergoing routine cervical screening by cytology status prior to implementation of an HPV vaccination program
2014, Journal of Clinical Virology
Citation Excerpt :
Briefly, 10 μl 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 μl of DNA) detecting mucosal HPV types, as previously described [21] Ultimately, DNA testing positive by AMP or the in-house PCR were genotyped (50 μl 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.
Data on the prevalence of cervical HPV genotypes in Australia by age and by grade of cytological abnormality are sparse.
Measure prevalence of HPV genotypes among 2620 Australian women by age and cytology status.
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.
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 <21 y to 5.8% in >40 y) and vaccine-targeted genotypes (HPV 6/11/16/18) (34.1% in <21 y to 2.4% 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.
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.

View all citing articles on Scopus

View full text

Short communicationHuman papillomavirus genotyping using a modified linear array detection protocol

Abstract

Section snippets

Acknowledgements

Virology

J. Clin. Virol.

Virology

The causal relation between human papillomavirus and cervical cancer

J. Clin. Pathol.

Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis

Br. J. Cancer

Human papillomavirus type specific DNA and RNA persistence—implications for cervical disease progression and monitoring

J. Med. Virol.

Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method

J. Clin. Microbiol.

Short communication
Human papillomavirus genotyping using a modified linear array detection protocol