Heather Cubie

Management of women who test positive for high-risk types of human papillomavirus: the HART study

BACKGROUND Certain types of human papillomavirus (HPV) are the primary cause of almost all cervical cancers. HPV testing of cervical smears is more sensitive but less specific than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN2+). HPV testing as a primary screening approach requires efficient management of HPV-positive women with negative or borderline cytology. We aimed to compare the detection rate and positive predictive values of HPV assay with cytology and to determine the best management strategy for HPV-positive women. METHODS We did a multicentre screening study of 11085 women aged 30-60 years. Women with borderline cytology and women positive for high-risk HPV with negative cytology were randomised to immediate colposcopy or to surveillance by repeat HPV testing, cytology, and colposcopy at 12 months. FINDINGS HPV testing was more sensitive than borderline or worse cytology (97.1% vs 76.6%, p=0.002) but less specific (93.3% vs 95.8%, p<0.0001) for detecting CIN2+. Of 825 randomised women, surveillance at 12 months was as effective as immediate colposcopy. In women positive for HPV at baseline, who had surveillance, 73 (45%) of 164 women with negative cytology and eight (35%) of 23 women with borderline cytology were HPV negative at 6-12 months. No CIN2+ was found in these women, nor in women with an initial negative HPV test with borderline (n=211) or mild (32) cytology. INTERPRETATION HPV testing could be used for primary screening in women older than 30 years, with cytology used to triage HPV-positive women. HPV-positive women with normal or borderline cytology (about 6% of screened women) could be managed by repeat testing after 12 months. This approach could potentially improve detection rates of CIN2+ without increasing the colposcopy referral rate.

Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population

Aims: If human papillomavirus (HPV) testing is to be included within cervical screening programmes, the importance of multiple HPV infections in cervical neoplasia needs to be determined. This study investigated the diversity of multiple HPV types in a routine cervical screening population, and assessed associations with cervical neoplasia. Methods: Overall HPV prevalence, type specific prevalence, and extent of multiple infection were assessed in residual material from 3444 liquid based cytology samples, using real time GP5+/GP6+ polymerase chain reaction for screening and linear array assay for genotyping. HPV status was studied in relation to age and concurrent cytological evidence of dyskaryosis. Results: Twenty per cent of samples were HPV positive. HPV type diversity was broad, and multiple HPV infections occurred in half of the HPV positive samples. Younger women were significantly more likely to harbour multiple high risk HPV (HR-HPV) infections. Infections with multiple HR-HPV types were found in 3.4% of samples negative for neoplasia and in 33.3%, 41.8%, and 40.4% of samples with borderline, mild, or high grade dyskaryosis, respectively. Single HR-HPV infections were found in 4.9%, 38.6%, 45.0%, and 51.1% of negative, borderline, mild, or high grade dyskaryosis samples, respectively. Conclusions: Multiple HR-HPV infections were most prevalent in young women. Multiple HR-HPV infections were not more frequent in high grade than in low grade cervical neoplasia, reflecting common sexual transmission of multiple HR-HPV. Prospective cohort studies linking sequential loss or gain of HPV types with cytological analysis are required to assess the impact of multiple HR-HPV infections on neoplastic progression.

Introduction and sustained high coverage of the HPV bivalent vaccine leads to a reduction in prevalence of HPV 16/18 and closely related HPV types

Background:In 2008, a national human papillomavirus (HPV) immunisation programme began in Scotland for 12–13 year old females with a three-year catch-up campaign for those under the age of 18. Since 2008, three-dose uptake of bivalent vaccine in the routine cohort aged 12–13 has exceeded 90% annually, while in the catch-up cohort overall uptake is 66%.Methods:To monitor the impact of HPV immunisation, a programme of national surveillance was established (pre and post introduction) which included yearly sampling and HPV genotyping of women attending for cervical screening at age 20. By linking individual vaccination, screening and HPV testing records, we aim to determine the impact of the immunisation programme on circulating type-specific HPV infection particularly for four outcomes: (i) the vaccine types HPV 16 or 18 (ii) types considered to be associated with cross-protection: HPV 31, 33 or 45; (iii) all other high-risk types and (iv) any HPV.Results:From a total of 4679 samples tested, we demonstrate that three doses (n=1100) of bivalent vaccine are associated with a significant reduction in prevalence of HPV 16 and 18 from 29.8% (95% confidence interval 28.3, 31.3%) to 13.6% (95% confidence interval 11.7, 15.8%). The data also suggest cross-protection against HPV 31, 33 and 45. HPV 51 and 56 emerged as the most prevalent (10.5% and 9.6%, respectively) non-vaccine high-risk types in those vaccinated, but at lower rates than HPV 16 (25.9%) in those unvaccinated.Conclusions:This data demonstrate the positive impact of bivalent vaccination on the prevalence of HPV 16, 18, 31, 33 and 45 in the target population and is encouraging for countries which have achieved high-vaccine uptake.

Persistent high risk HPV infection associated with development of cervical neoplasia in a prospective population study

Aims: To monitor the association between the course of high risk human papillomavirus (HR-HPV) infection and the development of cervical neoplasia over time, from a baseline of normal cervical cytology. Methods: This paper presents the follow up data from a previous cross sectional analysis. Women from a screening population who had normal cytology and who were HR-HPV positive were recalled after two to three years for cytology and HPV genotyping. The development of cervical neoplasia at follow up was related to the course of HPV infection (clearance, persistence, or sequential infection) and the presence of single or multiple HPV infections at baseline. A comparator control group of women who were HPV and cytologically negative at baseline were selected from the same population. Results: Twelve cases of dyskaryosis were found in women who were HPV positive at baseline; four were high grade. Only three cases of low grade dyskaryosis were found in the control group. Women with type specific persistent infections were significantly more likely to develop cervical neoplasia than women who cleared the infection (p = 0.0001) or were sequentially infected with different types (p = 0.001). Women with multiple HPV infections at baseline were no more likely to develop cervical dyskaryosis than those with a single infection. Conclusions: Type specific persistent HR-HPV infection as monitored by genotyping can identify women at increased risk of cervical neoplasia more accurately than a single or repeated presence/absence HPV test. The cost effectiveness of such an approach should be investigated by an appropriate, large scale cost–benefit analysis.

Diseases associated with human papillomavirus infection

Human papillomaviruses (HPVs) are ubiquitous, well adapted to their host and cleverly sequestered away from immune responses. HPV infections can be productive, subclinical or latent in both skin and mucosa. The causal association of HPV with cervical cancer, and increasingly with rising numbers of squamous cell carcinomas at other sites in both men and women, is increasingly recognised, while the morbidity of cutaneous HPV lesions, particularly in the immunosuppressed population is also significant. This chapter sets out the range of infections and clinical manifestations of the consequences of infection and its persistence and describes why HPVs are both highly effective pathogens and carcinogens, challenging to eliminate.

Reduction of low- and high-grade cervical abnormalities associated with high uptake of the HPV bivalent vaccine in Scotland

Background:In Scotland, a national HPV immunisation programme began in 2008 for 12- to 13-year olds, with a catch-up campaign from 2008 to 2011 for those under the age of 18. To monitor the impact of HPV immunisation on cervical disease at the population level, a programme of national surveillance was established.Methods:We analysed colposcopy data from a cohort of women born between 1988 and 1992 who entered the Scottish Cervical Screening Programme (SCSP) and were aged 20–21 in 2008–2012.Results:By linking datasets from the SCSP and colposcopy services, we observed a significant reduction in diagnoses of cervical intraepithelial neoplasia 1 (CIN 1; RR 0.71, 95% CI 0.58 to 0.87; P=0.0008), CIN 2 (RR 0.5, 95% CI 0.4 to 0.63; P<0.0001) and CIN 3 (RR 0.45, 95% CI 0.35 to 0.58; P<0.0001) for women who received three doses of vaccine compared with unvaccinated women.Conclusions:To our knowledge, this is one of the first studies to show a reduction of low- and high-grade CIN associated with high uptake of the HPV bivalent vaccine at the population level. These data are very encouraging for countries that have achieved high HPV vaccine uptake.

Worldwide genomic diversity of the high-risk human papillomavirus types 31, 35, 52, and 58, four close relatives of human papillomavirus type 16

ABSTRACT Among the more than one hundred formally described human papillomavirus (HPV) types, 18 are referred to as high-risk HPV types due to their association with anogenital cancer. Despite pathogenic similarities, these types form three remotely related taxonomic groups. One of these groups is called HPV species 9 and is formed by HPV-16, the most common and best-studied type, together with HPV-31, -33, -35, -52, -58, and -67. Previous worldwide comparisons of HPV-16 samples showed about 2% nucleotide diversity between isolates, which were subsequently termed variants. The distribution of divergent variants has been found to correlate frequently with the geographic origin and the ethnicity of the infected patients and led to the concept of unique African, European, Asian, and Native American HPV-16 variants. In the current study, we address the question of whether geography and ethnicity also correlate with sequence variations found for HPV-31, -35, -52, and -58. This was done by sequencing the long control region in samples derived from Europe, Asia, and Africa, and from immigrant populations in North and South America. We observed maximal divergence between any two variants within each of these four HPV types ranging from 1.8 to 3.6% based on nucleotide exchanges and, occasionally, on insertions and deletions. Similar to the case with HPV-16, these mutations are not random but indicate a relationship between the variants in form of phylogenetic trees. An interesting example is presented by a 16-bp insert in select variants of HPV-35, which appears to have given rise to additional variants by nucleotide exchanges within the insert. All trees showed distinct phylogenetic topologies, ranging from dichotomic branching in the case of HPV-31 to star phylogenies of the other three types. No clear similarities between these types or between these types and HPV-16 exist. While variant branches in some types were specific for Europe, Africa, or East Asia, none of the four trees reflected human evolution and spread to the extent illustrated by HPV-16. One possible explanation is that the rare HPV types that we studied spread and thereby diversified more slowly than the more abundant HPV-16 and may have established much of todays variant diversity already before the worldwide spread of humans 100,000 years ago. Most variants had prototypic amino acid sequences within the E6 oncoprotein and a segment of the L1 capsid protein. Some had one, two, or three amino acid substitutions in these regions, which might indicate biological and pathogenic diversity between the variants of each HPV type.

Automation-assisted versus manual reading of cervical cytology (MAVARIC): a randomised controlled trial

BACKGROUND The standard for reading cervical cytology is for a cytoscreener to manually search across an entire slide for abnormal cells using a conventional microscope. Automated technology can select fields of view to assess abnormal cells, which allows targeted reading by cytoscreeners. In the Manual Assessment Versus Automated Reading In Cytology (MAVARIC) trial, we compared the accuracy of these techniques for the detection of underlying disease. METHODS For this randomised controlled trial, women aged 25-64 years undergoing primary cervical screening in Manchester, UK, were randomly assigned (1:2) to receive either manual reading only or paired reading (automation-assisted reading and manual reading), between March 1, 2006, and Feb 28, 2009. In the paired arm, two automated systems were used-the ThinPrep Imaging System and the FocalPoint GS Imaging System. General practices and community clinics were randomised to either ThinPrep or to SurePath (for the FocalPoint system) liquid-based cytology with block randomisation stratified by deprivation index. Samples were then individually randomised to manual reading only or paired reading only. Laboratory staff were unaware of the allocation of each slide and concealment was maintained until the end of the reporting process. The primary outcome was sensitivity of automation-assisted reading relative to manual reading for the detection of underlying cervical intraepithelial neoplasia grade 2 or worse (CIN2+) in the paired arm. This trial is registered, number ISRCTN66377374. FINDINGS 73,266 liquid-based cytology samples were obtained from women undergoing primary cervical screening; 24,688 allocated to the manual-only arm and 48,578 to the paired-reading arm. Automation-assisted reading was 8% less sensitive than manual reading (relative sensitivity 0·92, 95% CI 0·89-0·95), which was equivalent to an absolute reduction in sensitivity of 6·3%, assuming the sensitivity of manual reading to be 79%. Specificity of automation-assisted reading relative to manual reading increased by 0·6% (1·006, 95% CI 1·005-1·007). INTERPRETATION The inferior sensitivity of automation-assisted reading for the detection of CIN2+, combined with an inconsequential increase in specificity, suggests that automation-assisted reading cannot be recommended for primary cervical screening.

A longitudinal study of HPV detection and cervical pathology in HIV infected women

Objective: To monitor the presence and persistence of high risk (HR) human papillomavirus (HPV) in cervical brushings from HIV infected women. Methods: Prospective observational cohort study of HIV infected women. Women were enrolled from the cohort of 164 HIV infected women who attend the colposcopy clinic at the Edinburgh Regional Infectious Diseases Unit. A single cervical brush scrape was obtained from 39 women and two or more samples from 63 women who attended regularly at approximately 6 monthly intervals. HPV typing was carried out using a commercial hybrid capture assay (HCA). Details of antiretroviral therapy, cytological assessment, and histological evaluation were made available and the interrelation with HR-HPV detection analysed. Results: Abnormal cervical cytology, particularly of low grade, was common in these HIV infected women. HR-HPV types were detected in 25% of the women with normal cytology, while over 80% of those with abnormal cytology of any grade were HR-HPV positive. Persistent HR-HPV, as defined by two or more consecutive HPV positive results, was common and found in 27/63 women from whom multiple samples were obtained. HR-HPV was detected at high levels whether or not patients were receiving antiretroviral therapy. Profound immunosuppression was not necessarily associated with progression of cervical disease and no cases of invasive cervical disease were seen. Conclusion: While mild dyskaryosis (low grade squamous intraepithelial lesion (LSIL)) and persistence of HR-HPV are common in HIV infected women in Edinburgh, regular cytological and colposcopic evaluation with appropriate intervention and treatment appears to limit the progression of cervical disease.

Presence of antibodies to human papillomavirus virus-like particles (VLPs) in 11-13-year-old schoolgirls

To allow meaningful approaches to vaccine development, it is important to know the extent of exposure to human papillomavirus (HPV) within the general population, and particularly the age at which the at risk population is infected. The humoral response to human papillomavirus is directed largely to conformationally‐dependent epitopes on the whole virion. Virus‐like particles (VLPs) of HPV types 1, 2, and 16 were produced using a baculovirus expression system, and were used in the intact state as antigen in an indirect ELISA. Anonymised serum samples from a cohort of Edinburgh schoolgirls were tested for the presence of IgG antibodies directed against the VLPs. The reproducibility of the ELISA was assured by repeated testing of control samples, and by testing all samples in duplicate and, where possible, on several occasions. Of 1,192 tested with the HPV16 VLPs, 90 (7.6%) were classified as clearly positive, and a further 87 (7.3%) were positive but close to the cutoff calculated by comparison with a group of consistently negative sera. Antibodies to HPV2 were detected in 37.5% (407/1,139) and antibodies to HPV 1 in 51.9% (558/1,076) of the schoolgirls. Antibodies to both HPV1 and HPV2 were found frequently, being present in 29.7% (295/993) of samples tested; 40 samples had antibodies to all three types. The significance of these results is discussed. J. Med. Virol. 56:210–216, 1998.