A real-time PCR assay for HPV52 detection and viral load quantification.

BACKGROUND: Human papillomavirus (HPV) 52 is one of the most frequent high risk HPV types found in cervical and anal infections. Reliable and well characterized methods for HPV 52 detection are therefore of great importance. Unfortunately one of the most widely used commercially available HPV typing assays, the Roche Linear Array (LA), detects type 52 only through its XR probe which cross-reacts with types 33, 35 and 58 and fails to give unambiguous detection of HPV 52. METHODS: To address this problem a real-time TaqMan PCR assay for HPV 52 targeting the E6/E7 region was developed and validated, which can be applied as robust duplex assay simultaneously detecting beta-globin as genomic control and reference or as highly sensitive single target detection assay. RESULTS: Optimized primer and probe concentrations produced linear PCR amplifications over seven logs of targets (10(1) - 10(7)). The detection limit for HPV 52 was reproducibly at 10 copies per reaction for the duplex assay format and 5 copies for the single-plex format. The assay was very type-specific and no amplification signal was observed with 10(7) copies of the related HPV33, 35, and 58 DNA. Of 89 samples that tested unambiguously positive for HPV 52 in the LA, 75 were confirmed in the duplex format and 88 in the single-plex format. An additional 100 samples negative for HPV 52 in LA were all negative in both HPV 52 real-time PCR assay formats. CONCLUSIONS: These results indicate 92.6% and 99.5% accuracy relative to LA for the duplex and single-plex formats, respectively. In ongoing testing of 18,484 from various studies, 10.8% required the HPV52 TaqMan assay to unequivocally determine the status. Including the HPV 52 duplex assay provides the ability to monitor variations in the cell yield in various methods of sample collection and processing. This additional information is useful in QC monitoring of epidemiologic studies.

Human papillomavirus infection and cytologic abnormalities of the anus and cervix among HIV-infected women in the study to understand the natural history of HIV/AIDS in the era of effective therapy (the SUN study).

BACKGROUND: Human papillomavirus (HPV) infection of the cervix and related abnormal cervical cytology in HIV-infected women has been well described. Little is known about anal HPV infection in HIV-infected women. METHODS: The SUN Study is a prospective cohort study of 700 HIV-infected patients including 167 women. At baseline, patients completed a behavioral questionnaire and provided, among other samples, cervical and anal swabs for HPV detection and genotyping and for cytologic examination. Here, we present the available baseline data on the 167 women in the SUN study. RESULTS: Baseline results were available for 120 women (median age: 38 years, 57% non-Hispanic black, median CD4 cell count 444.5 cells/mm3), of whom, 77% were taking antiretroviral therapy. The prevalences in the anus and cervix of any HPV were 90% and 83%, respectively (P = 0.039), and of high-risk (HR) types 85% and 70%, respectively, (P = 0.001). There was no significant difference in the prevalences of abnormal cytology between the anus and cervix: 38% and 33%, respectively (P = 0.217). Although the presence of abnormal cervical cytology was associated with the presence of abnormal anal cytology (relative risk: 1.7, P = 0.024), its sensitivity (52.5%) and positive predictive value positive (45.6%) for identifying women with abnormal anal cytology were poor. A history of anal sex was not associated with anal HPV infection or abnormal anal cytology. CONCLUSIONS: In this cohort of HIV-infected women, anal HPV infection was more prevalent and diverse than cervical HPV infection. Anal cytologic abnormalities were as prevalent as cervical cytologic abnormalities, and although abnormal cervical cytology was predictive of abnormal anal cytology, results were not highly concordant. These data support the need for studies of anal cytologic screening of HIV-infected women.

Differences and changes in human papillomavirus type 16 variant status in human immunodeficiency virus-positive adults are not uncommon.

Human papillomavirus type 16 (HPV-16) genotype variants have been the subject of several investigations, but study participants have rarely been sampled more than once. In this study, among a cohort of human immunodeficiency virus (HIV)-infected adults, HPV-16 variants were investigated in samples collected concurrently from the anus and cervix, as well as in serial samples collected from the same anatomical site at 12-month intervals. HPV-16 variants in stored extracts of cervical and anal samples were determined from subjects with multiple visits and at least one sample positive for HPV-16. Seven polymorphic nucleotide positions within the E6 region were analysed by pyrosequencing to determine genotype variants. Of 364 samples examined, 176 anal and 39 cervical swabs from 84 different subjects yielded unequivocal sequences of eight major HPV-16 variants. Eight samples contained probable novel HPV-16 variants and in one sample two variants were detected. In eight out of 29 (27.6%) anal-cervical sample pairs positive for HPV-16, discordant variants were found. From 57 anal and nine cervical sample series of HPV-16-positive samples, a change in HPV-16 variant status over time was seen in nine (13.6%) instances (seven anal and two cervical) from eight different participants. Changes in HPV-16 variants in HIV-infected adults were seen most frequently when different anatomical sites were sampled, but were also observed over time.

Type-specific reproducibility of the Roche linear array HPV genotyping test.

BACKGROUND: Type-specific detection of human papillomavirus (HPV) is increasingly important for monitoring temporal and age-specific changes in type-specific prevalence in support of HPV vaccination efforts. The impact of sampling, extraction and assay characteristics on HPV results is increasingly recognized. Inter-assay comparability studies have been performed, but the robustness of type-specific results has neither been emphasized nor has the degree of intra-assay reproducibility been addressed. OBJECTIVES: Here we describe the general and type-specific reproducibility of the linear array HPV genotyping test (Roche Molecular Diagnostics, Indianapolis, IN). STUDY DESIGN: Extracts of 276 cervical samples from two ongoing epidemiologic HPV studies were retested while blinded to prior results. The testing involved five different reagent lots and three technologists. RESULTS: Concordance for HPV detection (sample positive versus negative for any of the 37 types) was high (98.2%, kappa=0.959). Type-specific concordance for individual HPV types was also high (99.4%, kappa=0.915), and most samples (83.0%) showed complete concordance for all types. CONCLUSIONS: Type-specific reproducibility of the linear array HPV genotyping test is good but not perfect. The results suggest that type-specific performance should be included in the evaluation of HPV typing formats.

Prevalence of HPV infection among females in the United States.

CONTEXT: Human papillomavirus (HPV) infection is estimated to be the most common sexually transmitted infection. Baseline population prevalence data for HPV infection in the United States before widespread availability of a prophylactic HPV vaccine would be useful. OBJECTIVE: To determine the prevalence of HPV among females in the United States. DESIGN, SETTING, AND PARTICIPANTS: The National Health and Nutrition Examination Survey (NHANES) uses a representative sample of the US noninstitutionalized civilian population. Females aged 14 to 59 years who were interviewed at home for NHANES 2003-2004 were examined in a mobile examination center and provided a self-collected vaginal swab specimen. Swabs were analyzed for HPV DNA by L1 consensus polymerase chain reaction followed by type-specific hybridization. Demographic and sexual behavior information was obtained from all participants. MAIN OUTCOME MEASURES: HPV prevalence by polymerase chain reaction. RESULTS: The overall HPV prevalence was 26.8% (95% confidence interval [CI], 23.3%-30.9%) among US females aged 14 to 59 years (n = 1921). HPV prevalence was 24.5% (95% CI, 19.6%-30.5%) among females aged 14 to 19 years, 44.8% (95% CI, 36.3%-55.3%) among women aged 20 to 24 years, 27.4% (95% CI, 21.9%-34.2%) among women aged 25 to 29 years, 27.5% (95% CI, 20.8%-36.4%) among women aged 30 to 39 years, 25.2% (95% CI, 19.7%-32.2%) among women aged 40 to 49 years, and 19.6% (95% CI, 14.3%-26.8%) among women aged 50 to 59 years. There was a statistically significant trend for increasing HPV prevalence with each year of age from 14 to 24 years (P<.001), followed by a gradual decline in prevalence through 59 years (P = .06). HPV vaccine types 6 and 11 (low-risk types) and 16 and 18 (high-risk types) were detected in 3.4% of female participants; HPV-6 was detected in 1.3% (95% CI, 0.8%-2.3%), HPV-11 in 0.1% (95% CI, 0.03%-0.3%), HPV-16 in 1.5% (95% CI, 0.9%-2.6%), and HPV-18 in 0.8% (95% CI, 0.4%-1.5%) of female participants. Independent risk factors for HPV detection were age, marital status, and increasing numbers of lifetime and recent sex partners. CONCLUSIONS: HPV is common among females in the United States. Our data indicate that the burden of prevalent HPV infection among females was greater than previous estimates and was highest among those aged 20 to 24 years. However, the prevalence of HPV vaccine types was relatively low.

Human papillomavirus type 16 variant assignment by pyrosequencing.

Polymorphisms in human papillomavirus type 16 (HPV16) result in variants from the prototype sequence which can be designated according to geographic distribution and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Detection of variants has been used to distinguish persistent HPV16 infection from re-infection in natural history studies, and variants have been associated with an increased risk of cervical disease in some populations. Variant determination usually relies on conventional Sanger sequencing of regions of the viral genome, with the major variant group assignments requiring the sequencing of only seven polymorphic sites spread over a 242-bp region of the E6 gene. We applied pyrosequencing to facilitate rapid sequencing and enable the simultaneous detection of multiple variants. A single-stranded template for pyrosequencing was prepared by amplifying a 314-bp fragment (nt 75-388) with a biotin at the 5'-end of the reverse primer to facilitate strand separation and purification. Polymorphisms at the nucleotide sites 109, 131, 132, 143, 145, 178 and 350 were determined in three separate sequencing reactions, one of which was a multiplex format. Pyrosequencing of 97 HPV16-positive exfoliated cervical samples confirmed the Sanger sequencing results; however pyrosequencing identified additional variants in several samples containing mixed variants.

Quantitation of site-specific HPV 16 DNA methylation by pyrosequencing.

Human papillomavirus (HPV) is a necessary but insufficient cause of cervical cancer. Factors influencing transcription, such as epigenetic silencing through viral DNA methylation, may impact neoplastic progression. Pyrosequencing technology was applied to quantify methylation at 19 cytosine guanine dinucleotide (CpG) sites in the L1 3' and long control region (LCR) of HPV 16 DNA using cell lines, CaSki ( approximately 400 integrated copies of HPV 16) and SiHa (1-2 integrated copies of HPV 16) that differ in their transcriptional activity. Methylation levels ranged from 20 to 100% in CaSki and from 0 to 85% in SiHa over the entire 19 CpG sites, with a >40-fold difference in the methylation levels of their promoter and enhancer regions (SiHa<2% and CaSki 79%). The method was successful at a limiting dilution of 1-4 HPV 16 DNA copies/3000 cells, a level compatible with most clinical samples. The results were not affected by fixation in methanol-based liquid cytology collection fluid or method of extraction. Conditions optimized with cell lines were applicable to fixed exfoliated cervical cells. Pyrosequencing provides a quantitative site-specific assessment of methylation at multiple CpG sites without cloning, and is thus suited to large-scale molecular epidemiologic studies.

Human papillomavirus in amniotic fluid.

BACKGROUND: There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes. METHODS: Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV. RESULTS: There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23-55 years. All samples were beta-globin positive. HPV was not detected in any of the paired samples. CONCLUSION: Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none.

Patterns of cellular and HPV 16 methylation as biomarkers for cervical neoplasia.

Aberrant promoter methylation of biologically relevant genes in cervical cancer and uneven CpG distribution within the human papillomavirus 16 (HPV 16) enhancer region have been reported. Cervical samples and questionnaires from 151 women screened for cervical cancer in Appalachian Ohio were analyzed. Methylation was measured by bisulfite sequencing in candidate gene sites in ESR1, DCC, p16, and LINE1 elements. Among 89 HPV 16-positive women, CpG sites in the E6 promoter and enhancer regions and the L1 region of the HPV 16 genome were measured. Methylation levels were compared by cervical cytology and HPV 16 status. HPV methylation was low regardless of cytology status, however E6 methylation was significantly higher in women with normal cytology. ESR1 and DCC methylation were significantly higher in HPV 16-positive women. Increased methylation at sites in the E6 promoter region was associated with lower odds of abnormal cytology. Increased methylation in candidate genes was associated with higher odds of abnormal cytology, particularly DCC region 2.4, DCC region 2.6, ESR1 region 3.2, and LINE1 site 1.2. HPV 16 genome CpG methylation was low except for the L1 region. In general, lower HPV 16 methylation and higher candidate gene methylation levels were associated with higher odds of abnormal cytology.

Impact of HPV assay on observed population prevalence.

Type-specific surveillance of human papillomavirus (HPV) has been proposed as an early indicator of vaccine impact. Longitudinal comparison of HPV typing results requires stable assays with high type-specific reproducibility. Assays are evolving and the impact of even minor changes in the assay format may be difficult to anticipate. We initiated a population-based study of HPV with the prototype line blot (PLB) assay. These reagents were replaced by the research use only Linear Array (LA) HPV Genotyping kit. The assays are similar in principle and earlier comparisons found increased sensitivity and detection of more types per sample with LA; however, in samples from women with cervical abnormalities, the overall concordance was good. Slight changes in sensitivity may be more significant in samples from a general population with lower viral loads in the samples. Residual extracts from 3001 self-collected vaginal swabs from women in the general US population originally tested with PLB were retested with LA. With LA, all the samples were hybridized. PLB hybridization was restricted to samples with probable amplicon in gel electrophoresis. For HPV detection, the agreement between the 2 assays was 78.6% (κ=0.55) with a positive concordance of 52.8%. However, this masks the observation that repeat testing with LA led to the detection of HPV in nearly twice as many samples. Agreement improves if comparison was restricted to the samples hybridized. These results emphasize that assay comparisons should consider the clinical-epidemiologic context of sample collection. Studies designed to examine temporal trends in type-specific prevalence should archive residual material to permit retesting if assays change.

Anogenital human papillomavirus in sexually abused and nonabused children: a multicenter study.

OBJECTIVES: To characterize the epidemiology of genital human papillomavirus (HPV) infection in children without previous consensual sexual activity, comparing HPV prevalence by certainty of child sexual abuse (CSA). PATIENTS AND METHODS: Patients presenting for evaluation of CSA in 8 sites in Atlanta, Houston, Harrisburg, and New York City were recruited along with patients presenting for unrelated health visits. CSA certainty was classified as definite, probable, possible, or no evidence following published guidelines and the results of history, physical examination, and laboratory tests. Urine and swabs of external genitalia were tested for HPV using L1 consensus polymerase chain reaction. RESULTS: The study included 576 participants (89.9% female) aged 6 months to 13 years (mean: 7.9); 534 of whom were evaluated for CSA and 42 for unrelated reasons. Of those evaluated for CSA, 14 had genital warts. One or more HPV types were detected in 11.8% (61 of 517) of participants with adequate samples. HPV detection was more likely among abused participants (definite, probable, or possible) than among participants without evidence of CSA (13.7% and 1.3%, respectively; P < .0001) and increased with certainty of abuse (8.4%, 15.6%, and 14.5% in participants with possible, probable, and definite CSA, respectively; P < .0001). Participants aged 10 years or older had a higher prevalence of HPV (20.6%) than others (5.6%) (P < .0001). CSA, anogenital warts, and age were independently associated with HPV detection. CONCLUSIONS: HPV detection was associated with CSA and increased with CSA certainty. In this population, genital HPV seemed to behave as a sexually transmitted infection.

Simultaneous amplification and identification of 25 human papillomavirus types with Templex technology.

The majority of existing human papillomavirus (HPV) genotyping assays are based on multiplex PCR using consensus or degenerate primers. We developed a Templex HPV assay that simultaneously detects and identifies 25 common HPV genotypes in a single-tube reaction using type-specific primers for the HPV-specific E6 and E7 genes. The analytical sensitivities of the Templex assay for HPV type 16 (HPV-16), -18, and -56 were 20, 100, and 20 copies per reaction mixture, respectively. The Templex assay provides semiquantitative information on each type when multiple HPV types coexist in one reaction. We tested 109 clinical cervical specimens previously evaluated with the Digene HC2 high-risk HPV DNA test and found 95.4% concordance between the assay results. The Templex assay provided type-specific results and found multiple types in 29.2% (14 of 48) of high-risk HPV-positive samples. The entire Templex procedure, including DNA extraction, can be completed within 5 hours, providing a rapid and reliable diagnostic tool for HPV detection and typing that is amenable to automation.

Human papillomavirus type 16 E2 and E6/E7 variants.

OBJECTIVES: Polymorphisms in human papillomavirus (HPV) type 16 have been shown to be related to geographic areas and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Certain variants have been reported as being more likely to cause cervical disease; our objectives were to identify new HPV16 polymorphisms, to determine the linkage of the E2 and E6/E7 regions and to determine the minimum sequence necessary to classify variants. METHODS: We sequenced the complete E2, E6, and E7 regions in all HPV16-positive cervical samples identified in a case-control study of pre-invasive cervical disease. RESULTS: In the 100 samples analyzed, only one new polymorphism was identified, a synonymous change, T3205A, in region E2. The frequency distribution of variants in the sample set was 37 European prototypes and 27 E-G350, 16 AA, 5 Af1, 2 Af2, 8 E-C109G, 3 E-G131G, and 2 As. As shown by others, region E7 varied much less than E6 and E2. CONCLUSIONS: In each case, E2 changes were linked to the expected E6/E7 changes, and there was no evidence for recombination. The linkage between E2 and E6/E7 allows variant classification to be based on a short E6 sequence (nt 109-350).

Epidemiologic and viral factors associated with cervical neoplasia in HPV-16-positive women.

While infection with high-risk HPV is the most important risk factor for cervical cancer, HPV alone is insufficient. Our purpose was to identify viral and epidemiologic factors associated with cervical disease in HPV-16 DNA-positive women referred to colposcopy. We used a standardized interview to collect epidemiologic data from consenting women. Total nucleic acids from exfoliated cervical cells were used for all viral assays (HPV detection and typing using L1 consensus PCR with line probe hybridization, variant classification by sequencing, viral load and transcript copy determination by quantitative PCR and transcript pattern by nested RT-PCR). Cervical disease was based on colposcopic biopsy. Logistic regression was used to calculate ORs with 95% CIs. There were 115 HPV-16 positive women among 839 enrollees. By univariate analyses, age >25 years (OR = 3.05, 95% CI 1.20-7.76), smoking (OR = 3.0, 95% CI 1.19-7.56), high viral load (OR = 5.27, 95% CI 2.05-13.60), detection of both E6 and E6*I transcripts (OR = 10.0, 95% CI 2.1-47.58) and high transcript copies (OR = 5.56, 95% CI 2.05-13.60) were significant risk factors for CIN III with reference to No CIN/CIN I. Less than a third of the women (31.5%) had prototype HPV-16 detected, and variants showed no association with disease, viral load or transcription. Viral DNA and transcript copies were highly correlated, and the ratio of transcript copies to DNA copies was not changed with disease status. While viral load, transcript copies and transcript pattern were statistically associated with CIN III, none of these measures effectively discriminated between HPV-16 women with disease requiring treatment and those who could be followed. Cellular proliferation and differentiation pathways affected by HPV should be investigated as biomarkers for cervical cancer screening.

A case-control study of human papillomavirus and cervical squamous intraepithelial lesions (SIL) in Harris County, Texas: differences among racial/ethnic groups

Foi realizado um estudo caso-controle para analisar a associaçäo entre lesöes intra-epiteliais escamosas do colo uterino (SIL) e HPV entre mulheres brancas, negras e latinas em Harris County, Texas. Os casos foram identificados na M. D. Anderson Cancer Center Colposcopy Clinic, e os controles foram obtidos realizando-se exame de Papanicolau em duas clínicas do Departamento de Saúde. O HPV foi detectado por meio de ensaio de PCR (primer MY09/MY11). Foram construídos modelos de regressäo logística para estimar as odds ratios ajustadas (AOR), e seus intervalos de confiança de 95 por cento (IC) de SIL entre os grupos étnicos e graus da doença. A prevalência de HPV nas SIL de baixo grau (LSIL) foi de 64 por cento; nas de alto grau (HSIL), 84 por cento; e 19 por cento nos controles. O risco de SIL foi maior em mulheres latinas que em brancas e negras, sendo observadas, respectivamente, as seguintes AOR: 29,5 (12,4-70,5); 15,3 (6,0-33,8); e 5,8 (2,6-12,6). De forma similar, foram observadas diferenças para ambos LSIL (AOR, respectivamente, de 16,6; 7,7 e 4,3) e HSIL (AOR de 78,6; 34,6 e 142). Os resultados apóiam a existência de associaçäo entre SIL e HPV, diferenças na força de associaçäo com SILs e HSILs, e sugerem risco mais elevado para mulheres latinas e menor para mulheres negras.