Hélène Voyer

Use of PGMY Primers in L1 Consensus PCR Improves Detection of Human Papillomavirus DNA in Genital Samples

ABSTRACT 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.

Enhanced Detection and Typing of Human Papillomavirus (HPV) DNA in Anogenital Samples with PGMY Primers and the Linear Array HPV Genotyping Test

ABSTRACT 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.

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

ABSTRACT 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.

Absence of prolonged immunosilent infection with human immunodeficiency virus in individuals with high-risk behaviors

PURPOSE The presence in some individuals of a prolonged phase of infection with human immunodeficiency virus type 1 (HIV-1) before seroconversion remains controversial. This study was undertaken to determine with a sensitive in vitro amplification technique, the polymerase chain reaction (PCR), whether seronegative individuals with high-risk behaviors could harbor HIV-1 sequences in their peripheral blood mononuclear cells (PBMCs) and remain seronegative for more than 6 months. PATIENTS AND METHODS Seronegative individuals who engaged in unprotected anogenital intercourse with HIV-1-infected partners or with more than 10 individuals per year, and seronegative individuals who shared needles with seropositive partners, were recruited prospectively over 18 months. HIV-1 DNA and RNA sequences were detected in PBMCs of these individuals with three PCR assays using SK38/SK39, SK145/SK431, and SK68/SK69. Seronegative but PCR-positive patients were also evaluated with p24 antigen capture assay, radioimmunoprecipitation assay, and Western blot. The latter patients were followed prospectively to reproduce PCR-positive results and monitor serologic responses. RESULTS Sixty-one men and 18 women, with an average age of 34.1 +/- 7.6 years, were recruited: 56 were homosexual men, 18 were heterosexual women, and 5 were heterosexual men. Amplification reactions for HIV-1 of 104 PBMC specimens from 79 patients with negative or indeterminate serologies revealed that 4 patients (5.1%) were positive with PCR for HIV-1 DNA and RNA at the time of enrollment. Positive amplification reactions could not be reproduced in prospective samples for one patient. The analysis of a variable human genomic locus in this patients PBMCs demonstrated that the first PCR-positive sample and following PCR-negative samples originated from different patients, suggesting a specimen mix-up. Two of the three PCR-positive seronegative patients had symptoms suggestive of acute retroviral disease. Sera from all three patients contained p24 antigen. Two patients seroconverted within 1 month whereas one patient could not be followed prospectively. CONCLUSION Prolonged infection with HIV-1 without seroconversion was not found in our population of patients at very high risk for HIV-1 infection. All PCR-positive patients seroconverted in less than 1 month.

Estimating HPV DNA Deposition Between Sexual Partners Using HPV Concordance, Y Chromosome DNA Detection, and Self-reported Sexual Behaviors

Background Detection of human papillomavirus (HPV) DNA in genital samples may not always represent true infections but may be depositions from infected sexual partners. We examined whether sexual risk factors and a biomarker (Y chromosome DNA) were associated with genital HPV partner concordance and estimated the fraction of HPV detections potentially attributable to partner deposition. Methods The HITCH study enrolled young women attending a university or college in Montréal, Canada, and their male partners, from 2005 to 2010. We tested baseline genital samples for Y chromosome DNA and HPV DNA using polymerase chain reaction. Results Type-specific HPV concordance was 42.4% in partnerships where at least one partner was HPV DNA positive. Y chromosome DNA predicted type-specific HPV concordance in univariate analyses, but in multivariable models the independent predictors of concordance were days since last vaginal sex (26.5% higher concordance 0-1 vs 8-14 days after last vaginal sex) and condom use (22.6% higher concordance in never vs always users). We estimated that 14.1% (95% confidence interval [CI], 6.3-21.9%) of HPV DNA detections in genital samples were attributable to vaginal sex in the past week. Conclusions A substantial proportion of HPV DNA detections may be depositions due to recent unprotected vaginal sex.