Cervical cancer screening - The challenges of complete pathways of care in low-income countries: Focus on Malawi.

Cervical cancer is the fourth most common cancer among women globally, with approximately 580,000 new diagnoses in 2018. Approximately, 90% of deaths from this disease occur in low- and middle-income countries, especially in areas of high HIV prevalence, and largely due to limited prevention and screening opportunities and scarce treatment options. In this overview, we describe the opportunities and challenges faced in many low- and middle-income countries in delivery of cervical cancer detection, treatment and complete pathways of care. In particular, drawing on our experience and that of colleagues, we describe cervical screening and pathways of care provision in Malawi, as a case study of a low-resource country with high incidence and mortality rates of cervical cancer. Screening methods such as cytology - although widely used in high-income countries - have limited relevance in many low-resource settings. The World Health Organization recommends screening using human papillomavirus testing wherever possible; however, although human papillomavirus primary testing is more sensitive and detects precancers and cancers earlier than cytology, there are currently costs, infrastructure considerations and specificity issues that limit its use in low- and middle-income countries. The World Health Organization accepts the alternative screening approach of visual inspection with acetic acid as part of 'screen and treat' programmes as a simple and inexpensive test that can be undertaken by trained health workers and hence give wider screening coverage; however, subjectivity and variability in interpretation of findings between providers raise issues of false positives and overtreatment. Cryotherapy using either nitrous oxide or carbon dioxide is an established treatment for precancerous lesions within 'screen and treat' programmes; more recently, thermal ablation has been recognized as suitable to low-resource settings due to lightweight equipment, short treatment times, and hand-held battery-operated and solar-powered models. For larger lesions and cancers, complete clinical pathways (including loop excision, surgery, radiotherapy, chemotherapy and palliative care) are required for optimal care of women. However, provision of each of these components of cancer control is often limited due to limited infrastructure and lack of trained personnel. Hence, global initiatives to reduce cervical mortality need to adopt a holistic approach to health systems strengthening.

HPV infection and pre-term birth: a data-linkage study using Scottish Health Data.

Background: We aimed to investigate whether infection with high-risk (HR) types of human papilloma virus (HPV) or HPV-associated cervical disease were associated with preterm birth (<37 weeks gestation). In a sub-group of younger women who were eligible for the HPV vaccine, we aimed to determine whether prior vaccination against the specific HPV-types, HPV-16 and -18 modified preterm birth risk. Methods: This was a data-linkage study, which linked HPV-associated viral and pathological information (from the Scottish HPV Archive) from women aged 16-45 years to routinely collected NHS maternity- and hospital-admission records from 1999-2015. Pregnancy outcomes from 5,598 women with term live birth (≥37 weeks gestation, n=4,942), preterm birth (<37 weeks gestation, n=386) or early miscarriage (<13 weeks gestation, n=270). Of these, data from HPV vaccine-eligible women (n=3,611, aged 16-25 years) were available, of whom 588 had been vaccinated. HPV-associated disease status was defined as: HR HPV-positive no disease, low-grade abnormalities or high-grade disease. Results: High-grade HPV-associated cervical disease was associated with preterm birth (odds ratio=1.843 [95% confidence interval 1.101-3.083], p=0.020) in adjusted binary logistic regression analysis, in all women, but there were no associations with HR HPV-infection alone or with low-grade abnormalities. No associations between any HPV parameter and preterm birth were seen in vaccine-eligible women, nor was there any effect of prior vaccination. Conclusions: HPV-associated high-grade cervical disease was associated with preterm birth, but there were no associations with HR HPV-infection or low-grade cervical disease. Thus HPV-infection alone (in the absence of cervical disease) does not appear to be an independent risk factor for preterm birth. For women who have undergone treatment for CIN and become pregnant, these results demonstrate the need to monitor for signs of preterm birth.

Development of an in-house ELISA to detect anti-HPV16-L1 antibodies in serum and dried blood spots.

Measuring anti-HPV antibody levels is important for surveillance of the immunological response to both natural infection and vaccination. Here, an ELISA test for measurement of HPV-16L1 antibodies was developed and validated in sera and dried blood spots. An in-house ELISA was developed for measuring anti-HPV-16L1 IgA and IgG levels. The assay was standardized against WHO international standard serum and validated on serum, dried blood spots and cervical liquid based cytology samples from women attending colposcopy clinics in Scotland. Antibody avidity index was also measured in serum samples. The average HPV 16-L1 specific IgG and IgA levels measured in sera, in women attending a routine colposcopy service were 7.3 units/ml and 8.1 units/ml respectively. Significant correlations between serum and dried blood spot eluates for both IgG and IgA were observed indicating that the latter serve as a credible proxy for antibody levels. Average IgG Avidity Index was 35% (95% CI 25%-45%) suggesting previous, historical challenge with natural infection. This ELISA has potential for use in epidemiological and field studies of antibody prevalence and if coupled with avidity measurement may be of use in individual case monitoring of vaccine responses and failures.

Host chemokine signature as a biomarker for the detection of pre-cancerous cervical lesions.

BACKGROUND: The ability to distinguish which hrHPV infections predispose to significant disease is ever more pressing as a result of the increasing move to hrHPV testing for primary cervical screening. A risk-stratifier or "triage" of infection should ideally be objective and suitable for automation given the scale of screening. RESULTS: CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL12 emerged as the strongest, candidate biomarkers to detect underlying disease [cervical intraepithelial neoplasia grade 2 or worse (CIN2+)]. For CIN2+, CCL2 had the highest area under the curve (AUC) of 0.722 with a specificity of 82%. A combined biomarker panel of six chemokines CCL2, CCL3, CCL4, CXCL1, CXCL8, and CXCL12 provides a sensitivity of 71% and specificity of 67%. CONCLUSION: The present work demonstrates that the levels of five chemokine-proteins are indicative of underlying disease. We demonstrate technical feasibility and promising clinical performance of a chemokine-based biomarker panel, equivalent to that of other triage options. Further assessment in longitudinal series is now warranted. METHODS: A panel of 31 chemokines were investigated for expression in routinely taken archived and prospective cervical liquid based cytology (LBC) samples using Human Chemokine Proteomic Array kit. Nine chemokines were further validated using Procartaplex assay on the Luminex platform.

Eurogin roadmap 2017: Triage strategies for the management of HPV-positive women in cervical screening programs.

Cervical cancer screening will rely, increasingly, on HPV testing as a primary screen. The requirement for triage tests which can delineate clinically significant infection is thus prescient. In this EUROGIN 2017 roadmap, justification behind the most evidenced triages is outlined, as are challenges for implementation. Cytology is the triage with the most follow-up data; the existence of an HR-HPV-positive, cytology-negative group presents a challenge and retesting intervals for this group (and choice of retest) require careful consideration. Furthermore, cytology relies on subjective skills and while adjunctive dual-staining with p16/Ki67 can mitigate inter-operator/-site disparities, clinician-taken samples are required. Comparatively, genotyping and methylation markers are objective and are applicable to self-taken samples, offering logistical advantages including in low and middle income settings. However, genotyping may have diminishing returns in immunised populations and type(s) included must balance absolute risk for disease to avoid low specificity. While viral and cellular methylation markers show promise, more prospective data are needed in addition to refinements in automation. Looking forward, systems that detect multiple targets concurrently such as next generation sequencing platforms will inform the development of triage tools. Additionally, multistep triage strategies may be beneficial provided they do not create complex, unmanageable pathways. Inevitably, the balance of risk to cost(s) will be key in decision making, although defining an acceptable risk will likely differ between settings. Finally, given the significant changes to cervical screening and the variety of triage strategies, appropriate education of both health care providers and the public is essential.

Changes in the prevalence of human papillomavirus following a national bivalent human papillomavirus vaccination programme in Scotland: a 7-year cross-sectional study.

BACKGROUND: On Sept 1, 2008, Scotland launched routine vaccination for human papillomavirus (HPV) types 16 and 18, targeted at 12-13-year-old girls, of whom 92·4% were fully vaccinated in 2008-09. In this study, we report on vaccine effectiveness of the bivalent vaccine in these vaccinated women who attended for routine cervical screening at age 20-21 years. METHODS: In this 7-year cross-sectional study (covering birth cohorts 1988-1995), we sampled approximately 1000 samples per year from those attending cervical screening at age 20-21 years and tested each for HPV. By linkage to vaccination records we ascertained prevalence by birth cohort and vaccination status. Estimates of vaccine effectiveness for HPV types 16 and 18, HPV types 31, 33, and 45, other high-risk types, and any HPV were calculated using logistic regression. FINDINGS: In total, 8584 samples were HPV genotyped. Prevalence of HPV types 16 and 18 reduced substantially from 30·0% (95% CI 26·9-33·1) in the 1988 cohort to 4·5% (3·5-5·7) in the 1995 cohort, giving a vaccine effectiveness of 89·1% (85·1-92·3) for those vaccinated at age 12-13 years. All cross-protective types showed significant vaccine effectiveness (HPV type 31, 93·8% [95% CI 83·8-98·5]; HPV type 33, 79·1% [64·2-89·0]; HPV type 45, 82·6% [61·5-93·9]). Unvaccinated individuals born in 1995 had a reduced odds of HPV types 16 and 18 infection compared with those born in 1988 (adjusted odds ratio 0·13 [95% CI 0·06-0·28]) and reduced odds of HPV types 31, 33, and 45 (odds ratio 0·45 [0·23-0·89]). INTERPRETATION: Bivalent vaccination has led to a startling reduction in vaccine and cross-protective HPV types 7 years after vaccination. There is also evidence of herd protection against the vaccine-specific and cross-protective types in unvaccinated individuals born in 1995. These findings should be considered in cost-effectiveness models informing vaccine choice and models to shape the future of cervical screening programmes. FUNDING: Scottish Government and Chief Scientists Office.

HPV prevalence in women attending cervical screening in rural Malawi using the cartridge-based Xpert® HPV assay.

BACKGROUND AND OBJECTIVES: Early experience with Cepheid Xpert® HPV assay (Xpert® HPV) suggests that its quick turnaround time and ease of application might make it a relevant contender for routine use in low and middle income countries (LMICs). In the context of a cervical screening service in rural Malawi, we aimed to assess practicalities of local laboratory testing with Xpert® HPV and provide preliminary high-risk HPV (HR-HPV) prevalence data. STUDY DESIGN: Liquid-based cytology (LBC) specimens were collected from women attending cervical screening clinics in Nkhoma, Malawi. Xpert® HPV testing was carried out according to manufacturer's instructions. Partial genotyping results were obtained immediately (HPV 16, 18/45 and HR-HPV 'other'). Review of individual channel data provided further breakdown of other HR-HPV types into HPV 31 and related; HPV 51/59 and HPV 39 and related. RESULTS: Valid HR-HPV results were obtained from 750/763 samples. Most samples were from previously unscreened women, with 92.3% aged between 20 and 60 years. Overall HR-HPV positivity was 19.9%, with HR-HPV 'other' being more than twice as frequent as HPV 16 or HPV 18/45 and HPV 31-related (HPV 31, 33, 35, 52 or 58) most prevalent. Known HIV status was low (7.3%), but HR-HPV positivity in this group was much higher (43.4%). CONCLUSIONS: HR-HPV testing using Xpert® HPV was practical in a small rural laboratory. The rapid turnaround (within 2h) could facilitate a 'see and treat' programme. Partial genotyping allows assessment of risk beyond HPV 16/18. The high prevalence of HPV 31 and related types warrants further investigation.

Human Papilloma Virus (HPV) Oral Prevalence in Scotland (HOPSCOTCH): A Feasibility Study in Dental Settings.

The purpose of this study was to test the feasibility of undertaking a full population investigation into the prevalence, incidence, and persistence of oral Human Papilloma Virus (HPV) in Scotland via dental settings. Male and female patients aged 16-69 years were recruited by Research Nurses in 3 primary care and dental outreach teaching centres and 2 General Dental Practices (GDPs), and by Dental Care Teams in 2 further GDPs. Participants completed a questionnaire (via an online tablet computer or paper) with socioeconomic, lifestyle, and sexual history items; and were followed up at 6-months for further questionnaire through appointment or post/online. Saline oral gargle/rinse samples, collected at baseline and follow-up, were subject to molecular HPV genotyping centrally. 1213 dental patients were approached and 402 individuals consented (participation rate 33.1%). 390 completed the baseline questionnaire and 380 provided a baseline oral specimen. Follow-up rate was 61.6% at 6 months. While recruitment was no different in Research Nurse vs Dental Care Team models the Nurse model ensured more rapid recruitment. There were relatively few missing responses in the questionnaire and high levels of disclosure of risk behaviours (99% answered some of the sexual history questions). Data linkage of participant data to routine health records including HPV vaccination data was successful with 99.1% matching. Oral rinse/gargle sample collection and subsequent HPV testing was feasible. Preliminary analyses found over 95% of samples to be valid for molecular HPV detection prevalence of oral HPV infection of 5.5% (95%CI 3.7, 8.3). It is feasible to recruit and follow-up dental patients largely representative / reflective of the wider population, suggesting it would be possible to undertake a study to investigate the prevalence, incidence, and determinants of oral HPV infection in dental settings.

HPV-Related Oropharynx Cancer in the United Kingdom: An Evolution in the Understanding of Disease Etiology.

A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom. Archival tumor tissue blocks from 1,602 patients previously diagnosed with OPSCC (2002-2011) were collated from 11 centers. HPV status was determined with three validated commercial tests to provide valid data for 1,474 cases in total. Corresponding national incidence data from the same decade were obtained from UK Cancer registries. The overall proportion of HPV+ OPSCC between 2002 and 2011 was 51.8% [95% confidence interval (CI), 49.3-54.4], and this remained unchanged throughout the decade [unadjusted RR = 1.00 (95% CI, 0.99-1.02)]. However, over the same period, the incidence of OPSCC in the broader UK population underwent a 2-fold increase [age-standardized rate 2002: 2.1 (95% CI, 1.9-2.2); 2011: 4.1 (95% CI, 4.0-4.3)]. Although the number of OPSCCs diagnosed within the United Kingdom from 2002 to 2011 nearly doubled, the proportion of HPV+ cases remained static at approximately 50%. Our results argue that the rapidly increasing incidence of OPSCC in the United Kingdom cannot be solely attributable to the influence of HPV. The parallel increase in HPV+ and HPV- cases we documented warrants further investigation, so that appropriate future prevention strategies for both types of disease can be implemented. Cancer Res; 76(22); 6598-606. ©2016 AACR.

Clinical sensitivity of HPV assays for the detection of high grade cervical disease in cervical samples treated with glacial acetic acid.

BACKGROUND: Lysis of bloody liquid based cytology (LBC) specimens with glacial acetic acid (GAA) is performed to aid cytological interpretation. However, the influence of GAA treatment on HPV detection is not fully understood and in studies designed to assess this, few cases of high-grade disease have been included. OBJECTIVES: To assess the sensitivity of HPV molecular tests for the detection of high grade cervical disease in GAA treated samples STUDY DESIGN: A total of 207 specimens associated with high grade dyskaryosis and treated with GAA were collated prospectively. Overall 140 specimens had underlying CIN2+, including 88 CIN3. All specimens were tested with the Abbott RealTime High Risk HPV test (rtHPV) and the Qiagen Hybrid Capture 2High Risk HPV DNA test (HC2). Specimens associated with a CIN2+ that were negative by either assay were genotyped. RESULTS: The sensitivity of rtHPV for CIN2+ and CIN3+ was 92.8% (87.2, 96.5) and 94.3% (87.2, 98.1) respectively. Sensitivity of the HC2 for CIN2+ and CIN3+ was 97.2% (92.8, 99.2) and 96.6% (90.3, 99.2) respectively. The sensitivity of both assays in GAA treated specimens was thus consistent with the level required for clinical application. HPV negative, CIN2+ specimens were generally attributable to HPV types outside the explicit analytical range of the assays. CONCLUSIONS: The data indicate that GAA treatment has little impact on the detection of CIN2+ by HPV testing in LBC specimens.

Use of thermo-coagulation as an alternative treatment modality in a 'screen-and-treat' programme of cervical screening in rural Malawi.

The incidence of cervical cancer in Malawi is the highest in the world and projected to increase in the absence of interventions. Although government policy supports screening using visual inspection with acetic acid (VIA), screening provision is limited due to lack of infrastructure, trained personnel, and the cost and availability of gas for cryotherapy. Recently, thermo-coagulation has been acknowledged as a safe and acceptable procedure suitable for low-resource settings. We introduced thermo-coagulation for treatment of VIA-positive lesions as an alternative to cryotherapy within a cervical screening service based on VIA, coupled with appropriate, sustainable pathways of care for women with high-grade lesions and cancers. Detailed planning was undertaken for VIA clinics, and approvals were obtained from the Ministry of Health, Regional and Village Chiefs. Educational resources were developed. Thermo-coagulators were introduced into hospital and health centre settings, with theoretical and practical training in safe use and maintenance of equipment. A total of 7,088 previously unscreened women attended VIA clinics between October 2013 and March 2015. Screening clinics were held daily in the hospital and weekly in the health centres. Overall, VIA positivity was 6.1%. Almost 90% received same day treatment in the hospital setting, and 3- to 6-month cure rates of more than 90% are observed. Thermo-coagulation proved feasible and acceptable in this setting. Effective implementation requires comprehensive training and provider support, ongoing competency assessment, quality assurance and improvement audit. Thermo-coagulation offers an effective alternative to cryotherapy and encouraged VIA screening of many more women.

Use of HPV testing for cervical screening in vaccinated women--Insights from the SHEVa (Scottish HPV Prevalence in Vaccinated Women) study.

The management of cervical disease is changing worldwide as a result of HPV vaccination and the increasing use of HPV testing for cervical screening. However, the impact of vaccination on the performance of HPV based screening strategies is unknown. The SHEVa (Scottish HPV Prevalence in Vaccinated women) projects are designed to gain insight into the impact of vaccination on the performance of clinically validated HPV assays. Samples collated from women attending for first cervical smear who had been vaccinated as part of a national "catch-up" programme were tested with three clinically validated HPV assays (2 DNA and 1 RNA). Overall HR-HPV and type specific positivity was assessed in total population and according to underlying cytology and compared to a demographically equivalent group of unvaccinated women. HPV prevalence was significantly lower in vaccinated women and was influenced by assay-type, reducing by 23-25% for the DNA based assays and 32% for the RNA assay (p = 0.0008). All assays showed over 75% reduction of HPV16 and/or 18 (p < 0.0001) whereas the prevalence of non 16/18 HR-HPV was not significantly different in vaccinated vs unvaccinated women. In women with low grade abnormalities, the proportion associated with non 16/18 HR-HPV was significantly higher in vaccinated women (p < 0.0001). Clinically validated HPV assays are affected differentially when applied to vaccinated women, dependent on assay chemistry. The increased proportion of non HPV16/18 infections may have implications for clinical performance, consequently, longitudinal studies linking HPV status to disease outcomes in vaccinated women are warranted.

Prevalence of and Risk Factors for Anal Oncogenic Human Papillomavirus Infection Among HIV-Infected Women in France in the Combination Antiretroviral Therapy Era.

BACKGROUND: Little is known about the type-specific prevalence of anal human papillomavirus (HPV) infection and risk factors for anal high-risk (HR) HPV infection in human immunodeficiency virus (HIV)-infected women. METHODS: A cross-sectional study of anal and cervical HPV infection was nested within a gynecological cohort of HIV-infected women. Specimens were tested for type-specific DNA using a polymerase chain reaction-based assay. RESULTS: The study population consisted of 311 women with a median age of 45.3 years, of whom 42.8% originated from sub-Saharan Africa and 96.8% were receiving combination antiretroviral therapy. The median CD4(+)cell count was 612/µL, and the HIV load was <50 copies/mL in 84.1%. HR-HPV types were detected in the anal canal in 148 women (47.6%) and in the cervix in 82 (26.4%). HPV-16 was the most prevalent type in both the anal canal (13.2% of women) and the cervix (5.1%). In multivariable analysis, factors associated with prevalent anal HR-HPV infection were CD4(+)count <350/µL (odds ratio, 2.9; 95% confidence interval, 1.3-6.5), concurrent cervical lesions (2.6; 1.0-4.3), and cervical HR-HPV infection (1.8; 1.0-3.2). CONCLUSIONS: The high prevalence of HR-HPV types, including HPV-16, in the anal canal of HIV-positive women is concerning. Anal cancer screening should be considered for HIV-positive women as part of their routine care.

High Prevalence of Anal Human Papillomavirus-Associated Cancer Precursors in a Contemporary Cohort of Asymptomatic HIV-Infected Women.

BACKGROUND: Although human immunodeficiency virus (HIV)-infected women are at high risk for anal cancer, few data have been published on prevalence of and risk factors for anal precancer and potential screening strategies in this risk group. METHODS: A cross-sectional anal screening study was nested in a gynecological cohort of HIV-infected women. Anal swab specimens were collected for cytology and human papillomavirus (HPV) testing. High-resolution anoscopy, with biopsy when indicated, was systematically performed. RESULTS: Among the 171 enrolled women, median age was 47.3 years and 98% were receiving combination antiretroviral therapy. Median CD4(+) count was 655 cells/µL and HIV load was <50 copies/mL in 89% of subjects. High-grade anal intraepithelial neoplasia or worse (HG-AIN+) was diagnosed in 12.9% (n = 21). In multivariable analysis, a history of cervical squamous intraepithelial lesion (odds ratio [OR], 4.2; 95% confidence interval [CI], 1.1-16.4) and anal HPV-16 infection (OR, 16.1; 95% CI, 5.4-48.3) was associated with increased risk of HG-AIN+. Abnormal anal cytology and HPV-16 infection performed best as a screening strategy for HG-AIN+ histology, with positive likelihood ratios of 3.4 (95% CI, 2.3-5.1) and 4.7 (95% CI, 2.5-8.7) and negative likelihood ratios of 0.2 (95% CI, .07-.8) and 0.4 (95% CI, .2-.9), respectively. CONCLUSIONS: HIV-infected women with a history of HPV-associated cervical disease are at increased risk for HG-AIN+ and should be offered anal cancer screening. Anal cytology and HPV-16 genotyping had the best screening performance. Anal cytology is easy to perform routinely; it may be the best candidate for screening for HG-AIN among HIV-infected women.

Increased cycling cell numbers and stem cell associated proteins as potential biomarkers for high grade human papillomavirus+ve pre-neoplastic cervical disease.

High risk (oncogenic) human papillomavirus (HPV) infection causes cervical cancer. Infections are common but most clear naturally. Persistent infection can progress to cancer. Pre-neoplastic disease (cervical intraepithelial neoplasia/CIN) is classified by histology (CIN1-3) according to severity. Cervical abnormalities are screened for by cytology and/or detection of high risk HPV but both methods are imperfect for prediction of which women need treatment. There is a need to understand the host virus interactions that lead to different disease outcomes and to develop biomarker tests for accurate triage of infected women. As cancer is increasingly presumed to develop from proliferative, tumour initiating, cancer stem cells (CSCs), and as other oncogenic viruses induce stem cell associated gene expression, we evaluated whether presence of mRNA (detected by qRT-PCR) or proteins (detected by flow cytometry and antibody based proteomic microarray) from stem cell associated genes and/or increased cell proliferation (detected by flow cytometry) could be detected in well-characterised, routinely collected cervical samples from high risk HPV+ve women. Both cytology and histology results were available for most samples with moderate to high grade abnormality. We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3. SOX2, TP63 and human gonadotrophin mRNAs were upregulated in high grade disease. Immunohistochemistry showed that SOX2 and TP63 proteins clearly delineated tumour cells in invasive squamous cervical cancer. Samples from women with CIN3 showed increased proliferating cells. We believe that these markers may be of use to develop triage tests for women with high grade cervical abnormality to distinguish those who may progress to cancer from those who may be treated more conservatively.

Clinical performance of RNA and DNA based HPV testing in a colposcopy setting: Influence of assay target, cut off and age.

BACKGROUND: As HPV testing is used increasingly for cervical disease management, there is a demand to optimise the performance of HPV tests, particularly with respect to specificity. OBJECTIVES: To compare the clinical performance of an HPV DNA and a RNA based test in women with cytological abnormalities. The influence of age and assay cut off on test performance was also assessed. STUDY DESIGN: A prospective comparison of the Hybrid Capture 2 test (HC2) and the Aptima HPV assay (AHPV) was performed within a colposcopy setting. Clinical sensitivity and specificity were determined for the detection of cervical intraepithelial neoplasia (CIN) grade 2 or worse. RESULTS: Both assays were >90% sensitive for the detection of CIN2+. AHPV was slightly more specific than HC2 [49.9% (46.8-53.1) vs 45.9% (42.8, 49.1), p<0.0001]. Raising HC2 cut off to 2 RLU did not improve specificity. A cut-off of 10 RLU increased specificity by approximately 10% - although this led to a reduction in sensitivity of 6.3% which equated to 24 missed cases of CIN2+. Both assays were more specific in women over 30 years of age, compared to women under 30 (p<0.001). CONCLUSION: Although AHPV was more specific than HC2 in the total cohort (p<0.001), we found this difference to be smaller than other studies. This could be attributed to different indications for colposcopic referral across different settings. This study also confirms the relatively poor specificity of commercial HPV assays in women under 30.

Assessing the detection of human papillomavirus late mRNA in liquid base cytology samples for risk stratification of cervical disease.

Molecular human papillomavirus (HPV) testing is an important and developing tool for cervical disease management. However there is a requirement to develop new HPV tests that can differentiate between clinically significant and benign, clinically insignificant infection. Evidence would indicate that clinically significant infection is linked to an abortive HPV replication cycle. In particular the later stages of the replication cycle (i.e., production of late messenger (m) RNAs and proteins) appear compromised. Compared to current DNA-based tests which indicate only presence or absence of virus, detecting virus mRNAs by reverse transcriptase PCR (RT-PCR) may give a more refined insight into viral activity and by implication, clinical relevance. A novel quantitative (q)RT-PCR assay was developed for the detection of mRNAs produced late in the viral replication cycle. Initially this was validated on HPV-containing cell lines before being applied to a panel of 223 clinical cervical samples representing the cervical disease spectrum (normal to high grade). Samples were also tested by a commercial assay which detects expression of early HPV E6/E7 oncoprotein mRNAs. Late mRNAs were found in samples associated with no, low and high grade disease and did not risk-stratify HPV infection. The data reveal hidden complexities within the virus replication cycle and associated lesion development. This suggests that future mRNA tests for cervical disease may require quantitative detection of specific novel viral mRNAs.

Estimation of HPV prevalence in young women in Scotland; monitoring of future vaccine impact.

BACKGROUND: Estimation of pre-immunisation prevalence of HPV and distribution of HPV types is fundamental to understanding the subsequent impact of HPV vaccination. We describe the type specific prevalence of HPV in females aged 20-21 in Scotland who attended or defaulted from cervical screening using three specimen types; from attenders liquid based cytology and from defaulters urine or self-taken swabs. METHODS: Residual liquid based cytology samples (n = 2148), collected from women aged 20-21 attending for their first smear were genotyped for HPV. A sample (n = 709) from women who had defaulted from screening was also made available for HPV testing through the use of postal testing kits (either urine samples (n = 378) or self-taken swabs (n = 331)). Estimates of prevalence weighted by deprivation, and for the postal testing kit, also by reminder status and specimen type were calculated for each HPV type. The distribution of HPV types were compared between specimen types and the occurrence of multiple high-risk infections examined. The influence of demographic factors on high-risk HPV positivity and multiple infections was examined via logistic regression. RESULTS: The prevalence of any HPV in young women aged 20-21 was 32.2% for urine, 39.5% for self-taken swab, and 49.4% for LBC specimens. Infection with vaccine specific types (HPV 16, 18) or those associated with cross-protection (HPV 31, 33, 45, 51) was common. Individuals were more likely to test positive for high-risk HPV if they resided in an area of high deprivation or in a rural area. The overall distribution of HPV types did not vary between defaulters and attenders. Multiple infections occurred in 48.1% of high-risk HPV positive individuals. Excluding vaccine types the most common pairing was HPV 56 and 66. CONCLUSIONS: Understanding of the pre-immunisation prevalence of HPV in young women puts Scotland in a prime position to assess the early effect of vaccination as the first highly vaccinated cohorts of individuals enter the screening programme. Differences in results with different specimen types must be taken into account when monitoring the impact of vaccination programmes.

Cervical cancers associated with human papillomavirus types 16, 18 and 45 are diagnosed in younger women than cancers associated with other types: a cross-sectional observational study in Wales and Scotland (UK).

BACKGROUND: Most cervical cancers are attributable to infection with one of fourteen types of human papillomavirus (HPV), but HPV types differ in oncogenic potential. Characterisation of cancers associated with specific HPV types is required to predict the likely impact of current prophylactic vaccines and the potential benefits of vaccine formulations including additional HPV types. OBJECTIVE: The study aimed to correlate HPV type with histology and age at diagnosis, in Invasive Cervical Cancers (ICCs) from two of the devolved countries of the UK (Wales and Scotland). STUDY DESIGN: Centralised histopathology review and rigorously standardised HPV-DNA typing were applied to 592 ICC diagnosed 2001-2006. HPV status was analysed in relation to histology and age at diagnosis. RESULTS: HPV infection was confirmed in 535/592 cases. Among the 497 tumours infected with single HPV types, the three most common types were HPV16 (62% 95%CI: 57.6-66.1), HPV18 (18.9% 95%CI: 15.7-22.6) and HPV45 (5.4% 95%CI: 3.7-7.8). HPV16 or 18 were present in 80.9% of HPV positive cases. Women with tumours associated with HPV types 16, 18 and 45 were on average 10.5 years younger at diagnosis than women with tumours associated with other HPV types. CONCLUSIONS: Prophylactic vaccines targeting HPV16 and 18 could potentially prevent up to 80.9% of ICC in the populations investigated. Cancers associated with HPV16, 18 and 45 were diagnosed at younger ages, supporting the hypothesis of faster progression than for tumours caused by other HPV types.

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.