The vaginal metabolome and microbiota of cervical HPV-positive and HPV-negative women: a cross-sectional analysis.

OBJECTIVE: Characterise the vaginal metabolome of cervical HPV-infected and uninfected women. DESIGN: Cross-sectional. SETTING: The Center for Health Behavior Research at the University of Maryland School of Public Health. SAMPLE: Thirty-nine participants, 13 categorised as HPV-negative and 26 as HPV-positive (any genotype; HPV+ ), 14 of whom were positive with at least one high-risk HPV strain (hrHPV). METHOD: Self-collected mid-vaginal swabs were profiled for bacterial composition by 16S rRNA gene amplicon sequencing, metabolites by both gas and liquid chromatography mass spectrometry, and 37 types of HPV DNA. MAIN OUTCOME MEASURES: Metabolite abundances. RESULTS: Vaginal microbiota clustered into Community State Type (CST) I (Lactobacillus crispatus-dominated), CST III (Lactobacillus iners-dominated), and CST IV (low-Lactobacillus, 'molecular-BV'). HPV+ women had higher biogenic amine and phospholipid concentrations compared with HPV- women after adjustment for CST and cigarette smoking. Metabolomic profiles of HPV+ and HPV- women differed in strata of CST. In CST III, there were higher concentrations of biogenic amines and glycogen-related metabolites in HPV+ women than in HPV- women. In CST IV, there were lower concentrations of glutathione, glycogen, and phospholipid-related metabolites in HPV+ participants than in HPV- participants. Across all CSTs, women with hrHPV strains had lower concentrations of amino acids, lipids, and peptides compared with women who had only low-risk HPV (lrHPV). CONCLUSIONS: The vaginal metabolome of HPV+ women differed from HPV- women in terms of several metabolites, including biogenic amines, glutathione, and lipid-related metabolites. If the temporal relation between increased levels of reduced glutathione and oxidised glutathione and HPV incidence/persistence is confirmed in future studies, anti-oxidant therapies may be considered as a non-surgical HPV control intervention. TWEETABLE ABSTRACT: Metabolomics study: Vaginal microenvironment of HPV+ women may be informative for non-surgical interventions.

Human papillomavirus and the risk of Barrett's esophagus.

Human papillomavirus (HPV) is strongly associated with squamous esophageal cancer. The potential role of HPV in Barrett's esophagus (BE) has been examined but remains unclear. The aim of the study was to determine the prevalence of HPV in esophageal and gastric tissues obtained from patients with and without BE. We designed a cross-sectional study was conducted with prospective enrollment of eligible patients scheduled for esophagogastroduodenoscopy (EGD). All participants had biopsies of endoscopic BE, squamous-lined esophagus, and stomach. Immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded tissue was conducted using monoclonal antibodies. Polymerase chain reaction (PCR) for HPV was performed on DNA extracted from esophageal biopsies snapped frozen within 30 minutes after endoscopic capture. The Roche HPV Linear Array Assay with PGMY primers that has high sensitivity for detecting 37 types of HPV was used. A total of 127 subjects were included: 39 with definitive BE had IHC done on samples from non-dysplastic BE, squamous esophagus, gastric cardia, and gastric body; and 88 control patients without BE had IHC done on squamous esophageal samples, gastric cardia, and gastric body. HPV was not detected in any of the samples in either group. For confirmation, HPV DNA PCR was performed on randomly selected samples from 66 patients (both esophagus and BE from 13 patients with BE, and 53 esophagus from patients without BE); no sample had HPV DNA detected via PCR in the presence of adequate quality control. HPV infection does not play a role in the formation of non-dysplastic Barrett's esophagus in men in the United States.

Human papillomavirus prevalence, cervical abnormalities and risk factors among female sex workers in Lima, Peru.

Female sex workers (FSWs) are at high risk of human papillomavirus (HPV) infection. Questionnaires were administered to 200 FSWs aged 18-26 years in Lima, Peru, to gather risk behaviours, and cervical swab samples were collected for Pap smears and HPV DNA testing as part of a longitudinal study. Participants reported a median of 120 clients in the past month, and 99.2% reported using condoms with clients. The prevalence of any HPV in cervical samples was 66.8%; 34 (17.1%) participants had prevalent HPV 16 or 18, and 92 (46.2%) had one or more oncogenic types. Fifteen women had abnormal Pap smears, 13 of which were HPV DNA positive. Fewer years since first sex was associated with oncogenic HPV prevalence in a model adjusted for previous sexually transmitted infection (STI) status and condom use with partners (prevalence ratio = 0.77, 95% confidence interval [CI] = 0.60-0.97). Our data confirm the high rates of HPV transmission among FSWs in Peru, highlighting the need for early and effective strategies to prevent cervical cancer.

Oral sex practices, oral human papillomavirus and correlations between oral and cervical human papillomavirus prevalence among female sex workers in Lima, Peru.

Few data exist on oral human papillomavirus (HPV) prevalence in female sex workers (FSWs). Information regarding oral sex practices of 185 Peruvian FSWs, 18-26 years of age, was obtained via survey and compared with HPV testing results of oral rinse samples. Oral HPV prevalence was 14/185 (7.6%); four (28.9%) HPV genotypes were carcinogenic. One hundred and eighty-two participants reported having had oral sex; 95% reported condom use during oral sex with clients and 9.5% with partners. Women who had oral sex more than three times with their partners in the past month were more likely to have oral HPV than women who had oral sex three times or less (P = 0.06). Ten (71.4%) women with oral HPV were HPV-positive at the cervix; conversely 8.3% of women with cervical HPV were HPV-positive in the oral cavity. The prevalence of oral HPV was relatively low, considering the high rates of oral sex practiced by these women.

Kinetics of DNA load predict HPV 16 viral clearance.

INTRODUCTION: While high HPV 16 viral load measured at a single time point is associated with cervical disease outcomes, few studies have assessed changes in HPV 16 viral load on viral clearance. OBJECTIVE: To measure the association between changes in HPV 16 viral load and viral clearance in a cohort of Thai women infected with HPV 16. STUDY DESIGN: Fifty women (n=50) between the ages of 18-35 years enrolled in a prospective cohort study were followed up every three months for two years. Women positive for HPV 16 DNA by multiplex TaqMan assay at two or more study visits were selected for viral load quantitation using a type-specific TaqMan based real-time PCR assay. The strength of the association of change in viral load between two visits and viral clearance at the subsequent visit was assessed using a GEE model for binary outcomes. RESULTS: At study entry, HPV 16 viral load did not vary by infection outcome. A >2 log decline in viral load across two study visits was found to be strongly associated with viral clearance (AOR: 5.5, 95% CI: 1.4-21.3). HPV 16 viral load measured at a single time point was not associated with viral clearance. CONCLUSIONS: These results demonstrate that repeated measurement of HPV 16 viral load may be a useful predictor in determining the outcome of early endpoints of viral infection.

Prevalence and risk factors for carcinogenic human papillomavirus infections in rural Rakai, Uganda.

OBJECTIVE: To investigate self-administered vaginal swabs for assessing prevalence and correlates of carcinogenic human papillomavirus (HPV) infection in rural Rakai, Uganda. METHODS: 1003 sexually experienced women enrolled in a community cohort provided self-administered vaginal swabs collected at annual, home-based surveys. Carcinogenic HPV prevalence, adjusted odds ratios (AOR), 95% confidence intervals (CI) and associated risk factors were determined. RESULTS: Carcinogenic HPV prevalence was 19.2%: 46.6% among HIV positive and 14.8% among HIV negative women (p<0.001). Type-specific prevalence ranged from 2.0% (HPV 16 and 52) to 0.2% (HPV 31). Age-specific HPV prevalence decreased significantly (p<0.001) among HIV negative women; however, the decrease among HIV positive women was not as pronounced (p = 0.1). Factors independently associated with carcinogenic HPV infection were HIV (AOR 4.82, CI 3.10 to 7.53), age (AOR 4.97, 95% CI 2.19 to 11.26 for 15-19 year olds compared to 40+ years), more than two sex partners in the past year (AOR 2.21, CI 1.10 to 4.43) and self-reported herpes zoster, candidiasis or tuberculosis (AOR 4.52, CI 1.01 to 20.31). Married women were less likely to have prevalent carcinogenic HPV (AOR 0.46, CI 0.30 to 0.70). CONCLUSIONS: HPV prevalence and correlates measured using self-administered vaginal swabs were similar to studies that use cervical samples. Thus, self-collection can be used as a substitute for cervical specimens and provide an important tool for research in populations unwilling to undergo pelvic exam.

Prevalencia delvirus del papiloma humano (VPH) y otros factores de riesgo para el desarrollo de cáncer cervical en Guatemala / Prevalence of human papillomavirus (HPV) and other risk factors for the development of cervical cancer in Guatemala

El cáncer cervical es la malignidad ms común entre las mujeres del tercer mundo y en Guatemala, es responsable del 40% de todos los cánceres en ambos sexos y del 60% de todos los cánceres femeninos. La presencia de virus del Papiloma Humano (VPH) se considera entre otras, como factor de riesgo más importante para el desarrollo del cáncer cervical. El presente estudio fue llevado a cabo con el propósito de determinar la prevalencia de los tipos de VPH en un grupo de pacientes de Guatemala con cáncer cervical así como para evaluar los factores de riesgo asociados al desarrollo de la enfermedad. Con este propósito se llevó a cabo un estudio y controles en 112 pacientes con cáncer cervical invasivo (casos) y 102 mujeres sanas (controles). Se obtuvieron biopsias cervicales y se investigó la presencia de VPH usando dos métodos meleculares...

Human papillomavirus genotypes associated with cervical neoplasia in India.

The aim of this study was to determine the prevalence of human papillomavirus (HPV) genotypes in tissue with cervical neoplasias in patients from south and east India. Cross-sectional cervical tissue was obtained from 100 patients from south India and 30 patients from east India who had a biopsy for clinically invasive cervical cancer or cervical intraepithelial neoplasia (CIN) in a tertiary care hospital in India. DNA amplification was done with biotinylated PGMY 09/11 primers, and the line blot assay was used to determine the HPV genotype. HPV DNA was detected in 95% of invasive cancers (113/119) and 91% of CINs (10/11). A single HPV type was found in 100 women (77%), and mixed infections were found in 23 women (18%). HPV 16 (60%) and HPV 18 (14%) were the most frequent types, but 16 other types (26, 31, 33, 35, 42, 45, 51, 52, 53, 56, 58, 61, 62, 64, 81, and 82) were also identified. HPV 16 was present in 63% of patients from south India and in 50% of patients from east India, while HPV 18 was present in 12% and 20%, respectively. There are several high-risk HPV types associated with cervical neoplasias of which types 16 and 18 are the most common.

Determinants of genital human papillomavirus detection in a US population.

This study investigated the association of selected demographic and behavioral characteristics with the detection of low-risk, high-risk, and uncharacterized genital human papillomavirus (HPV) in women attending clinic for routine nonreferral gynecologic health care. Cervical specimens obtained from 3863 women 18-40 years old (mean, 28 years) with no history of high-grade cervical disease were analyzed for 38 HPV types. Overall, HPV prevalence was 39.2%. The prevalence of high-risk, low-risk, and uncharacterized HPV types was 26.7%, 14.7%, and 13.0%, respectively. As expected, the characteristics most strongly associated with overall HPV detection were age and numbers of lifetime and recent sex partners. Low-risk, high-risk, and uncharacterized HPV detection increased with increasing numbers of sex partners. There was a decline in high-risk and low-risk HPV detection with increasing age but little change in uncharacterized HPV detection. These results suggest that the uncharacterized HPV types have a different natural history than either low-risk or high-risk HPV types.

Associations between smoking and adenocarcinomas and squamous cell carcinomas of the uterine cervix (United States).

OBJECTIVES: Few studies of smoking and cervical carcinoma have addressed the rare cervical adenocarcinomas or used DNA-based tests to control for human papillomavirus (HPV) infection. METHODS: This multicenter case-control study included 124 adenocarcinoma cases, 307 community controls (matched on age, race, and residence to adenocarcinoma cases), and 139 squamous carcinoma cases (matched on age, diagnosis date, clinic, and disease stage to adenocarcinoma cases). Participants completed risk-factor interviews and volunteered cervical samples for PCR-based HPV testing. Polychotomous logistic regression generated adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for both histologic types. RESULTS: Eighteen percent of adenocarcinoma cases, 43% of squamous carcinoma cases, and 22% of controls were current smokers. After control for HPV and other questionnaire data, adenocarcinomas were consistently inversely associated with smoking (e.g. current: OR = 0.6, 95% CI 0.3-1.1; > or = 1 pack per day: OR = 0.7, 95% CI 0.4-1.3), while squamous carcinomas were positively associated with smoking (e.g. current: OR = 1.6, 95% CI 0.9-2.9; > or = 1 pack per day: OR = 1.8, 95% CI 1.0-3.3). Results in analyses restricted to HPV-positive controls were similar. CONCLUSION: Smoking has opposite associations with cervical adenocarcinomas and squamous carcinomas. Although both histologic types are caused by HPV and arise in the cervix, etiologic co-factors for these tumors may differ.

Evaluation of self-collected cervicovaginal cell samples for human papillomavirus testing by polymerase chain reaction.

As human papillomavirus (HPV) becomes accepted as the central cause of cervical cancer, longitudinal studies are shifting focus away from causality to a more detailed investigation of the natural history of HPV infections. These studies commonly require repeated samples for HPV testing over several years, usually collected during a pelvic exam, which is inconvenient to the participants and costly to the study. To alleviate the inconvenience and cost of repeated clinic visits, it has been proposed that women collect cervicovaginal cells themselves, hopefully increasing participation in the natural history studies. We evaluated the technical feasibility of self-collection of cervicovaginal cells using a Dacron swab for HPV DNA detection. We compared the self-collected swab sample and two clinician-administered swab samples (one from the endocervix and another from the ectocervix) from a total of 268 women participating in a case-control study of adenocarcinoma and squamous cell carcinomas of the uterine cervix (111 cases and 157 controls). HPV DNA was detected and genotyped using an L1 consensus PCR assay. The overall agreement between the clinician- and self-collected swabs was excellent [88.1%; kappa = 0.73 (95% confidence interval (CI), 0.61-0.85)]. The correlation was highest between the two clinician-administered swabs [kappa = 0.81 (95% CI, 0.69-0.93)] but was still excellent when comparing either clinician-administered swab to the self-administered sample [kappa = 0.75 (95% CI, 0.63-0.87) and 0.67 (95% CI, 0.55-0.79) for ectocervix and endocervix, respectively]. The type-specific agreement between samples was higher for high-risk, or cancer-associated, HPV genotypes than for low risk, noncancer-associated HPV genotypes when comparing the self-administered swab sample to the clinician-administered swab sample (kappa = 0.78 for high-risk versus 0.66 for low-risk HPV infections, t = -1.45, P = 0.15). The decrease in agreement for low risk types was largely attributable to an increased detection of these types in the self-administered sample (McNemar's chi2 = 6.25, P = 0.01 for clinician- versus self-administered swab comparisons). The agreement did not vary significantly by age, menopausal status, case status, or clinic center. We have demonstrated that a self-collected Dacron swab sample of cervicovaginal cells is a technically feasible alternative to clinician-administered cervical cell collection in natural history studies of HPV and cervical cancer.

A prospective study of human papillomavirus (HPV) type 16 DNA detection by polymerase chain reaction and its association with acquisition and persistence of other HPV types.

Human papillomavirus (HPV)-16 causes about half the cases of cervical cancer worldwide and is the focus of HPV vaccine development efforts. Systematic data are lacking as to whether the prevention of HPV-16 could affect the equilibrium of infection with other HPV types and thus alter the predicted impact of vaccination on the occurrence of cervical neoplasia. Therefore, the associations of HPV-16 detection with subsequent acquisition of other HPV types and with the persistence of concomitantly detected HPV types were examined prospectively among 1124 initially cytologically normal women. Preexisting HPV-16 was generally associated with an increased risk for subsequent acquisition of other types. HPV-16 did not affect the persistence of concomitant infections, regardless of type. These findings suggest that the prevention or removal of HPV-16 is not likely to promote the risk of infection with other types, a theoretical concern with current vaccination efforts.

Use of hormone replacement therapy and adenocarcinomas and squamous cell carcinomas of the uterine cervix.

INTRODUCTION: Exogenous hormones may influence the development of cervical adenocarcinomas. Incidence rates of adenocarcinomas and use of noncontraceptive hormones have increased since the 1970s, but few studies have investigated this potential relationship. METHODS: We conducted a multicenter case-control study of 124 women with adenocarcinomas, 139 women with squamous cell carcinomas matched on age, diagnosis date, clinic, and stage of disease (in situ or invasive) to adenocarcinoma cases, and 307 healthy community controls who were also matched on age, ethnicity, and residence to adenocarcinoma cases. Participants completed in-person interviews regarding exogenous hormone use before diagnosis and other risk factors and volunteered cervical samples for human papillomavirus (HPV) testing via a PCR-based method. Odds ratios (ORs) with 95% confidence intervals (CIs) estimated relative risks. RESULTS: Only 13 adenocarcinoma cases (10.5%), 7 squamous carcinoma cases (5%), and 20 controls (6.5%) had used noncontraceptive hormones for menopausal symptoms, irregular periods, or disease prevention; most use was short-term, former use. Ever-use was associated with adenocarcinomas (OR = 2.1, 95% CI 0.95-4.6) but not squamous carcinomas (OR = 0.85, 95% CI 0.34-2.1). No trends were seen with duration of use or ages at first use, but unopposed estrogens were positively associated with adenocarcinomas (OR = 2.7). Unopposed estrogens remained associated with adenocarcinomas (OR = 2.0) when analyses were restricted to the HPV-positive controls. Menopausal status was not associated with adenocarcinomas or squamous carcinomas and did not modify the other associations. CONCLUSION: Although small numbers warrant tentative conclusions, exogenous estrogens, especially unopposed estrogens, were positively associated with adenocarcinomas. Noncontraceptive hormones were negatively but weakly associated with squamous carcinomas.

Improved amplification of genital human papillomaviruses.

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.

Oral contraceptives as risk factors for cervical adenocarcinomas and squamous cell carcinomas.

To assess the hypothesis that oral contraceptives (OCs) increase the risk of cervical adenocarcinomas, we conducted a six-center case-control study of 124 patients with adenocarcinomas, 139 with squamous cell carcinomas, and 307 population controls. Women between the ages of 18 and 69 who were newly diagnosed with cervical adenocarcinomas between 1992 and 1996 were eligible. Healthy female controls and a second case group of incident cervical squamous cell carcinomas were matched to the adenocarcinoma cases. All participants were interviewed regarding OCs, other risk factors for cervical carcinoma, and utilization of cytological screening, and a PCR-based test determined HPV genotype of cervical samples for both case groups and controls. Use of OCs was positively and significantly associated with adenocarcinomas and positively but weakly associated with squamous cell carcinomas. Associations between OCs and invasive adenocarcinomas (n = 91), squamous cell carcinoma in situ (n = 48), and invasive squamous cell carcinomas (n = 91) disappeared after accounting for HPV infection, sexual history, and cytological screening, but a positive association remained between current use of OCs and cervical adenocarcinoma in situ (n = 33). This association persisted after stratification by screening and sexual history and after restriction according to HPV status, but small numbers made it difficult to exclude detection bias, selection bias, or residual confounding by HPV as potential explanations Current OC use was associated with cervical adenocarcinomas in situ, but we saw no other evidence that OCs independently increase the risk of cervical carcinomas.

Nonisotopic detection and typing of human papillomavirus DNA in genital samples by the line blot assay. The Canadian Women's HIV study group.

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for beta-globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43. 5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of beta-globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.

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

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/microliter) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.

Generation of entire human papillomavirus genomes by long PCR: frequency of errors produced during amplification.

Recently, several improvements of traditional PCR techniques have facilitated the amplification of significantly longer DNA target sequences. Here we report an improved method for amplification of entire human papillomavirus (HPV) genomes. Using rTth DNA polymerase, XL (Perkin-Elmer, Foster City CA), and the accompanying XL PCR buffer system, we have successfully amplified 8-kb genomes from approximately 10 copies of input reference strain HPV16 DNA. This long PCR (LPCR) method was subsequently used to amplify the entire HPV16 genome from clinical specimens. The fidelity with which the rTth DNA polymerase XL amplified target sequences under our chosen amplification conditions was estimated by partial sequencing of cloned LPCR products generated from cloned reference strain HPV16 genomes. A region spanning the HPV16 E6, E7, and part of the E1 open reading frames (ORFs) was sequenced in 29 clones. A total of 33 nucleotide substitutions were observed in the 23.5 kb sequenced. This corresponds to an error frequency of approximately one error per 700 bases. Finally, LPCR methods were used to amplify entire, novel HPV genomes from clinical specimens. LPCR primer pairs were designed for amplification of seven potentially novel HPV types. Amplicons of approximately 8 kb were generated from five of the seven HPV types targeted. One of the LPCR-generated novel genomes, CP141, was subsequently cloned and a partial sequence was determined.