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.

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.

High-risk HPV genotyping in the follow-up of women treated conservatively for microinvasive cervical cancer.

OBJECTIVES: Conservative management of women with microinvasive cervical cancer (International Federation of Gynecologists and Obstetricians stage IA) has led to prolonged and intensive cytological follow-up. We conducted a retrospective study to assess human papillomavirus status and genotypes at diagnosis and to find out whether there is an association between the persistence of high-risk human papillomavirus during follow-up and the detection of recurrent disease. STUDY DESIGN: Paraffin-embedded cervical biopsies in the pathology archives were identified from women with an initial large loop excision of the transformation zone or cone specimen diagnostic of microinvasive disease since 1991. RESULTS: We identified 45 women with a diagnosis of microinvasive cervical cancer. Human papillomavirus was detected in 87% of the initial diagnostic specimens. Human papillomavirus testing showed a negative predictive value of 100% for recurrent disease with a sensitivity of 100%. CONCLUSION: Human papillomavirus testing has an important role in the follow-up of women treated conservatively for stage IA cervical cancer.

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.

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.

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.

The role of human papillomavirus testing in cervical screening.

Organised, cytology-based cervical screening has led to a reduction in deaths associated with cervical cancer. Human papillomavirus (HPV) is necessary for the development of cervical cancer and associated pre-cursor cervical intraepithelial neoplasia and accumulated evidence suggests that incorporation of HPV testing could further refine screening programmes. HPV testing is discussed in the context of primary screening, for triage, and as a test of cure of treatment and possible value in developing countries. The high negative predictive value of a "double negative" cytology and HPV result could allow considerable changes in policy such as increased intervals between screening rounds, adjustment of age ranges for testing and schedule for return to routine screening post treatment. HPV testing for the triage of women to colposcopy with borderline or atypical squamous cells of undetermined significance (ASCUS) cytology could be clinically effective, but may be limited in women with low-grade squamous intraepithelial lesions (LSIL) or mild dyskaryosis by high HPV prevalence. Markers of HPV persistence harbour enormous potential to identify women at greatest risk of disease progression. Due to the diversity of existing cytology-based screening programmes, full cost-effectiveness analyses of HPV testing should be performed and assessed within local contexts.

The development of a quality assurance programme for HPV testing within the UK NHS cervical screening LBC/HPV studies.

BACKGROUND: Cervical screening by cytology is effective but lacks sensitivity. The addition of human papillomavirus (HPV) testing can improve the effectiveness of screening for early identification of cervical disease. As HPV testing represents a new technology, a quality assurance (QA) programme is necessary to confirm the accuracy of results. OBJECTIVE: Our main objective was to design a QA programme for use in the English NHS liquid-based cytology (LBC) and HPV Cervical Screening Pilot Study. Our second objective was to use the knowledge gained to design a QA scheme for future general use within cervical screening and HPV testing programmes. STUDY DESIGN: Four elements were included in the programme: provision of clinical samples of known HPV status for internal quality control (IQC), distribution of panels of unknown samples for external quality assessment (EQA), resubmission of aliquots of samples to the reference laboratory for repeat testing and resubmission to reference laboratory to check for transport problems. Three sites took part in the QA programme using PreservCyt medium and ThinPrep for LBC preparation. The assay used at test sites was HPV hybrid capture (hc2) while the quality assurance laboratory used a combination of hc2, in-house HPV polymerase chain reaction (PCR) tests and HPV linear array (LA). RESULTS: Four negative, three low positive and 11 positive pools were used in 22 distributions of IQC samples. Seven distributions each of five 'unknown' EQA samples were sent out. Over 400 samples underwent repeat testing. Discrepant samples were further assessed to provide an explanation. Inter- and intra-laboratory consistency was high as measured by Kappa statistics and 96% agreement for EQA samples was obtained. CONCLUSIONS: The validity of the QA programme was established and reproducibility in different lab settings was reassuring. These results support the use of hc2 as a potential screening test in diagnostic laboratories. The need for robust quality assurance of HPV testing in cervical screening programmes was confirmed and lessons learnt from this pilot study will be incorporated in future schemes.

Development of an automated extraction procedure for detection of human papillomavirus DNA in liquid based cytology samples.

Liquid based cytology samples are being used increasingly to improve cervical screening and have the advantage that residual cell suspension is available for other tests such as human papillomavirus (HPV) detection. However, as the transport medium is optimised for downstream cytology, problems can be experienced during extraction of nucleic acid. This study aimed to develop a robust protocol for automated extraction of HPV DNA from cervical, liquid based cytology samples using a high throughput robotic system. Considerable modification of existing clinical extraction protocols for swab specimens, together with optimisation of required sample input volume was required to reduce sample blockage during the extraction to acceptable levels. The blockage rate and optimal processing volume was assessed by extracting a fixed volume (1/4) of re-suspended material from the centrifuged pellets of 10, 5 and 1 ml aliquots of 200 specimens. Analysis revealed 17.5% blockage with specimens originating from 10 ml aliquots; 3% with 5 ml and no blockage with 1 ml aliquots of the same samples. A 3% blockage level is acceptable for an automatic well clearance procedure to be followed. HPV testing of the extracts by real-time PCR showed a 1.5% loss of sensitivity in extracts originating from 1 ml aliquots as compared with 5 ml aliquots with a consequent loss of detectable HPV genotypes after reverse hybridisation. In short, 5 ml of liquid based cytology specimen is recommended for nucleic acid extraction, to allow optimal detection of HPV types in clinical samples while retaining maximum efficiency of the robotic extraction procedure.

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.

External quality assessment for molecular detection of human papillomaviruses.

BACKGROUND: The United Kingdom National External Quality Assessment Service (UK NEQAS) distributes clinically relevant and educational specimens for external quality assessment (EQA). OBJECTIVES: The aim of this report was to assess the suitability of using liquid based cytology (LBC) samples for the EQA of molecular methods and to review the methods used by participants to detect the presence of high risk (HR) human papillomavirus (HPV) genotypes. STUDY DESIGN: Three pilot distributions were dispatched between January 2008 and January 2009 with each distribution consisting of four different specimens. RESULTS: Performance was good with over 90% of participants reporting correctly on the presence or absence of high risk genotypes in all but one specimen, specimen 9006 (82.1%). Specimen 9006 was a pooled specimen, negative for HR genotypes but containing low risk (LR) genotypes 61, 70 and 81. The most commonly used assay for the detection of the presence of HR HPV was the Digene Hybrid Capture II assay. The in-house PCR assays were most commonly associated with incorrect results, and the use of these assays decreased during the 13 month pilot study. CONCLUSIONS: The UK NEQAS molecular detection of HPV scheme provides a standardised, homogeneous and characterised clinical specimen, however this study has shown that genotyping results reported by participants were still varied. Inclusion of available HPV standards will help to standardise assays. Robust EQA of HPV molecular screening programmes will be essential for monitoring the impact of the HPV vaccine.

High-throughput detection of human papillomavirus-18 L1 gene methylation, a candidate biomarker for the progression of cervical neoplasia.

The L1 gene of human papillomavirus-18 (HPV-18) is consistently hypermethylated in cervical carcinomas, but frequently hypo- or unmethylated in exfoliated cells from asymptomatic patients. In precancerous lesions, L1 is sporadically hypermethylated, correlating with the severity of the neoplasia. In order to explore the potential of using L1 methylation as a workable biomarker for carcinogenic progression of HPV-18 infections in routinely taken samples, our aim was to develop methylation-detection techniques that were sensitive and rapid without being overly complex technically. Therein, we developed a methylation-specific PCR (MSP) through the design of primer sets that specifically amplify either methylated or unmethylated HPV-18 L1 DNA within bisulfite-modified sample DNA. Amplification of unmethylated and in vitro methylated HPV-18 DNA by MSP resulted in 2500 copies of either of the two L1 DNA species being detected, a satisfactory sensitivity considering that bisulfite treatment leads to the fragmentation of about 99% of sample DNA. The primers proved specific and did not generate false positive results at concentrations exceeding the lowest limit of detection by a factor of 400. DNA from carcinomas yielded PCR signals only with the methylation-specific primers, and not with primers specific for unmethylated L1 genes. The inverse result was obtained with DNA from precursor lesions that contained only hypomethylated DNA. High-grade precursor lesions and carcinomas that contained hyper- as well as hypomethylated L1 DNA yielded PCR signals with both primers. By developing a fluorescence based real-time PCR, we quantitatively analyzed samples with in vitro methylated and unmethylated L1 DNA, and could distinguish clinical samples with hyper- and hypomethylated DNA or mixtures of both DNAs. The methylation-specific and real-time PCR techniques permitted efficient HPV-18 L1 methylation analyses and open the door for larger-scale clinical studies where the utility of methylation status to predict the progression of HPV-18 infection and HPV-18 associated lesions is assessed.

Methylation of the human papillomavirus-18 L1 gene: a biomarker of neoplastic progression?

Epigenetic transcriptional regulation plays an important role in the life cycle of human papillomaviruses (HPVs) and the carcinogenic progression of anogenital HPV associated lesions. We performed a study designed to assess the methylation status of the HPV-18 genome, specifically of the late L1 gene, the adjacent long control region (LCR), and part of the E6 oncogene in cervical specimens with a range of pathological diagnoses. In asymptomatic infections and infections with precancerous (precursor) lesions, HPV-18 DNA was mostly unmethylated, with the exception of four samples where hypermethylation of L1 was detected. In contrast, L1 sequences were strongly methylated in all cervical carcinomas, while the LCR and E6 remained unmethylated. HeLa cells, derived from a cervical adenocarcinoma, contain chromosomally integrated HPV-18 genomes. We found that L1 is hypermethylated in these cells, while the LCR and E6 are unmethylated. Treatment of HeLa cells with the methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) led to the expected reduction of L1 methylation. After removal of 5-Aza-CdR, L1 methylation resumed and exceeded pretreatment levels. Unexpectedly, the LCR and E6 also became methylated under these conditions, albeit at lower levels than L1. We hypothesize that L1 is preferentially methylated after integration of the HPV genome into the cellular DNA, possibly since linearization prohibits its normal transcription, while the enhancer and promoter may be protected from methylation by transcription factors. Since our data suggest that HPV-18 L1 methylation can only be detected in carcinomas, except in some few precancerous lesions and asymptomatic infections, L1 methylation may constitute a powerful molecular marker for detecting this important step of neoplastic progression.

Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18.

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.

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

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 today's 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.

Papillomavirus subtypes are natural and old taxa: phylogeny of human papillomavirus types 44 and 55 and 68a and -b.

A human papillomavirus (HPV) type is defined as an HPV isolate whose L1 gene sequence is at least 10% different from that of any other type, while a subtype is 2 to 10% different from any HPV type. In order to analyze the phylogeny behind the subtype definition, we compared 49 isolates of HPV type 44 (HPV-44) and its subtype HPV-55, previously misclassified as a separate type, and 41 isolates of the subtype pair HPV-68a and -b, sampled from cohorts in four continents. The subtypes of each pair are separated by deep dichotomic branching, and three of the four subtypes have evolved large phylogenetic clusters of genomic variants forming a "star" phylogeny, with some branches specific for ethnically defined cohorts. We conclude that subtypes of HPV types are natural and old taxa, equivalent to types, which either diverged more recently than types or evolved more slowly.

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

In 2000, we monitored the course and persistence of human papillomavirus (HPV) infection in 54 women who were HPV positive and free of any cytological disease using HPV-DNA genotyping with a linear array assay (baseline). The impact of HPV infection on development of cervical cytological abnormality (dyskaryosis) was monitored by repeat HPV genotyping and cytological assessment 2 years later. Detection of mRNA transcripts of known HPV oncogenes E6 and E7 using NASBA methodology and specific molecular beacons for five common HPV types was also performed at both time points. A total of 11/54 (20%) women developed dyskaryosis after 2 years with 31/54 and 23/54 women exhibiting transient and persistent infections respectively, as monitored by DNA genotyping. Women who maintained type-specific persistent HPV infection were significantly more likely to develop dyskaryosis compared to those who exhibited a transient infection (P = 0.001). The presence of HPV mRNA E6/E7 transcripts was less sensitive but more specific for the detection of disease at follow up. Moreover, women who were DNA positive and also positive for mRNA transcripts at baseline were significantly more likely to harbour persistent infection compared to those in whom DNA only was detected at baseline (P = 0.013). This study highlights the importance of detecting persistent type specific HPV infection to identify those women more at risk of developing cervical abnormalities, either by repeated DNA genotyping, or potentially by RNA based techniques that may be more predictive of persistent infection if performed at a single time point.