The natural history of type-specific human papillomavirus infections in female university students.

Little is known about the average duration of type-specific human papillomavirus (HPV) infections and their patterns of persistence. The objectives of this study were to evaluate the rate of acquisition and clearance of specific HPV types in young women. Female university students (n = 621) in Montreal were followed for 24 months at 6-month intervals. At each visit, a cervical specimen was collected. HPV DNA was detected using the MY09/MY11 PCR protocol followed by typing for 27 HPV genotypes by a line blot assay. The Kaplan-Meier technique was used to estimate the cumulative probability of acquiring or clearing a HPV infection considering types individually or in high-risk (HR) or low-risk (LR) groups defined by oncogenic potential. Incidence rates were 14.0 cases/1000 women-months (95% confidence interval, 11.4-16.3) and 12.4 cases/1000 women-months (95% confidence interval, 10.4-14.8) for acquiring HR and LR HPV infections, respectively. The 24-month cumulative rates of acquisition were highest for HPV-16 (12%), HPV-51, and HPV-84 (8%). Of the incident infections, HPV-16 was the most persistent (mean duration, 18.3 months), followed by HPV-31 and HPV-53 (14.6 and 14.8 months, respectively). HPV-6 and HPV-84 had the shortest mean duration time (<10 months) The mean durations of incident, same-type LR or HR HPV infections were 13.4 months and 16.3 months, respectively. Whereas the majority of episodes with a type-specific HPV infection cleared within 2 years, there were many women who were either reinfected with a new HPV genotype or presumably experienced reactivation of their initial infection.

Real-time PCR assays using internal controls for quantitation of HPV-16 and beta-globin DNA in cervicovaginal lavages.

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and beta-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.

Confirmatory real-time PCR assay for human papillomavirus (HPV) type 52 infection in anogenital specimens screened for HPV infection with the linear array HPV genotyping test.

A novel real-time PCR assay for detection of human papillomavirus type 52 (HPV-52) DNA (RT-52) was evaluated on 265 anogenital samples. RT-52 had a sensitivity of 98.4% and a specificity of 100% compared to conventional HPV-52 typing assays, including hybridization of PGMY products with an HPV-52-specific probe and PCR sequencing of HPV-52 E6.

Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the Linear array HPV genotyping test.

The Roche PGMY primer-based research prototype line blot assay (PGMY-LB) is a convenient tool in epidemiological studies for the detection and typing of human papillomavirus (HPV) DNA. This assay has been optimized and is being commercialized as the Linear Array HPV genotyping test (LA-HPV). We assessed the agreement between LA-HPV and PGMY-LB for detection and typing of 37 HPV genotypes in 528 anogenital samples (236 anal, 146 physician-collected cervical, and 146 self-collected cervicovaginal swabs) obtained from human immunodeficiency virus-seropositive individuals (236 men and 146 women). HPV DNA was detected in 433 (82.0%) and 458 (86.7%) samples with PGMY-LB and LA-HPV (P = 0.047), respectively, for an excellent agreement of 93.8% (kappa = 0.76). Of the 17,094 HPV typing results, 16,562 (1,743 positive and 14,819 negative results) were concordant between tests (agreement = 96.9%; kappa = 0.76). The mean agreement between tests for each type was 96.4% +/- 2.4% (95% confidence interval [CI], 95.6% to 97.2%; range, 86% to 100%), for an excellent mean kappa value of 0.85 +/- 0.10 (95% CI, 0.82 to 0.87). However, detection rates for most HPV types were greater with LA-HPV. The mean number of types per sample detected by LA-HPV (4.2 +/- 3.4; 95% CI, 3.9 to 4.5; median, 3.0) was greater than that for PGMY-LB (3.4 +/- 3.0; 95% CI, 3.1 to 3.6; median, 2.0) (P < 0.001). The number of types detected in excess by LA-HPV in anal samples correlated with the number of types per sample (r = 0.49 +/- 0.06; P = 0.001) but not with patient age (r = 0.03 +/- 0.06; P = 0.57), CD4 cell counts (r = 0.06 +/- 0.06; P = 0.13), or the grade of anal disease (r = -0.11 +/- 0.06; P = 0.07). LA-HPV compared favorably with PGMY-LB but yielded higher detection rates for newer and well-known HPV types.

The p53 codon 72 polymorphism and risk of high-grade cervical intraepithelial neoplasia.

BACKGROUND: The Arg/Arg genotype versus Arg/Pro or Pro/Pro at codon 72 of the p53 gene has been implicated in increasing susceptibility of the cervix to human papillomavirus (HPV) infection and thus altering cancer risk. However, research on this topic has been contentious, which prompted us to carry out a case-control study in the Montreal area. METHODS: Cases were women with histologically-confirmed high-grade cervical intraepithelial neoplasia (HGCIN). Controls were women without a history of cervical abnormalities. From each woman, we obtained a cervical specimen for HPV testing and p53 genotyping, and a questionnaire was completed. DNA sequencing was used to minimize genotype misclassification. A subsample of specimens was also genotyped using the TaqMan assay. RESULTS: There were 357 cases and 760 controls recruited between February 2001 and December 2003. The distribution of Arg/Arg, Arg/Pro and Pro/Pro was 55.2, 36.4 and 8.4%, respectively, among cases, and 52.1, 38.7 and 9.2%, among controls, corresponding to an odds ratio (OR) adjusted for ancestral origin of 1.16 (95% confidence interval (CI): 0.9-1.5) for Arg/Arg versus other genotypes. When restricted to high-risk HPV-positive women, the adjusted ORs were 1.40 (CI: 0.9-2.1) and 2.12 (CI: 1.1-4.2), for Arg/Arg versus other genotypes and versus Pro/Pro, respectively. The findings were comparable with analyses of genotype results that agreed between DNA sequencing and TaqMan. CONCLUSIONS: In this study, we attempted to minimize selection bias, population stratification and genotype misclassification. The results suggest that the role of the p53 codon 72 polymorphism on HGCIN is weak at best. Further research may reveal if the polymorphism has a stronger influence on the risk of invasive cervical cancer.

Prevalence of selective inhibition of HPV-16 DNA amplification in cervicovaginal lavages.

HPV-16 viral load has been assessed with real-time PCR assays by measuring HPV-16 DNA and a human gene in genital samples. HPV-16 viral load measurements are thus based on the inference that inhibitors contained in samples will equally impede amplification of DNA sequences from HPV-16 and human DNA. We have previously shown that sample lysates can inhibit amplification of HPV-16 but not beta-globin DNA. In the current study, cervicovaginal lavages lysates considered adequate for PCR analysis by a qualitative beta-globin PCR test, were screened for the presence of inhibitors using internal controls (IC) for HPV-16 DNA and beta-globin in real-time PCR assays. Of 150 lysates screened with both ICs, 12 (8%) contained inhibitors. Inhibition of amplification of both ICs was demonstrated in four of these specimens. In eight lysates, amplification of HPV-16 IC only was impeded. Six (50%) of these 12 lysates tested positive for HPV-16 DNA despite the presence of PCR inhibitors. The HPV-16 viral load increased significantly after dilution of 11 of 12 lysates, demonstrating the presence of inhibitors in the undiluted lysate. Nine (90%) of 10 samples with inhibitors that were tested after dilution did not demonstrate inhibitory activity. The use of internal controls in real-time PCR is clearly essential to determine HPV viral loads since the effect of inhibitors on primer-driven genomic amplification is variable.

Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples.

The novel PGMY L1 consensus primer pair is more sensitive than the MY09 and MY11 primer mix for detection and typing with PCR of human papillomavirus (HPV) DNA in genital specimens. We assessed the diagnostic yield of PGMY primers for the detection and typing of HPV by comparing the results obtained with PGMY09/PGMY11 and MY09/MY11/HMB01 on 299 genital samples. Amplicons generated with PGMY primers were typed with the line blot assay (PGMY-line blot), while HPV amplicons obtained with the degenerate primer pool MY09/MY11/HMB01 were detected with type-specific radiolabeled probes in a dot blot assay (standard consensus PCR test). Cervicovaginal lavage samples (N = 272) and cervical scrape samples (N = 27) were tested in parallel with both PCR tests. The PGMY-line blot test detected the presence of HPV DNA more frequently than the standard consensus PCR assay. The concordance for HPV typing between the two assays was 84.3% (214 of 255 samples), for a good kappa value of 0.69. Of the 177 samples containing HPV DNA by at least one method, 40 samples contained at least one HPV type detected only with PGMY-line blot, whereas positivity exclusively with the standard consensus PCR test was found for only 7 samples (P < 0.001). HPV types 45 and 52 were especially more frequently detected with PGMY than MY primers. However, most HPV types were better amplified with PGMY primers, including HPV-16. Samples with discordant results between the two PCR assays more frequently contained multiple HPV types. Studies using PGMY instead of MY primers have the potential to report higher detection rates of HPV infection not only for newer HPV types but also for well-known genital types.