Prevalence of selective inhibition of HPV‐16 DNA amplification in cervicovaginal lavages

Abstract

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.

Human Papillomavirus Type 16 Viral Load Is Higher in Human Immunodeficiency Virus-Seropositive Women with High-Grade Squamous Intraepithelial Lesions Than in Those with Normal Cytology Smears

“…This is the first report on the role of HPV-16 viral load in cervical HPV infection and SIL in HIV-infected women combining real-time PCR and screening for PCR inhibition. The assay we used was demonstrated to be specific for HPV-16 and does not cross-react with other HPV types (6,7,14). Several studies reported a higher quantity of HPV DNA in HIVseropositive than HIV-seronegative women but did not evaluate the impact of HIV-induced immunosuppression on HPV viral load (5,11,12,16,18).…”

High Level of Correlation of Human Papillomavirus-16 DNA Viral Load Estimates Generated by Three Real-time PCR Assays Applied on Genital Specimens

“…One hundred forty-four HPV-16-positive samples (125 cervicovaginal lavages and 19 specimens collected using vaginal tampon) were selected for the current evaluation as described above. These samples had been screened in previous studies for the presence of inhibitors by real-time PCR using internal controls for HPV-16 and h-globin (28,31,32). Briefly, 1,000 copies of HPV-16 L1 internal control, 1,000 copies of HPV-16 E6 internal control, and 1,000 copies of h-globin internal control were amplified in separate capillaries with 2 AL specimen lysate and tested in a Light Cycler PCR and detection system (Roche Molecular Systems, Branchburg, NJ; refs.…”

“…Briefly, 1,000 copies of HPV-16 L1 internal control, 1,000 copies of HPV-16 E6 internal control, and 1,000 copies of h-globin internal control were amplified in separate capillaries with 2 AL specimen lysate and tested in a Light Cycler PCR and detection system (Roche Molecular Systems, Branchburg, NJ; refs. 28,31,32). The presence of PCR inhibitors was suspected when 1,000 copies of at least one of the internal controls generated a signal corresponding to <700 copies as explained in detail previously (31).…”

“…They attain levels of sensitivity near one copy per test and have been shown to be very specific (9,(31)(32)(33)(34)(35). Real-time HPV-16 PCR assays normalizing for total cell number have been developed for the investigation of evolution of HPV viral load in the natural history of CIN (8-10, 13, 15, 28, 33, 36-38).…”

“…Real-time HPV-16 PCR assays normalizing for total cell number have been developed for the investigation of evolution of HPV viral load in the natural history of CIN (8-10, 13, 15, 28, 33, 36-38). The intralaboratory and interlaboratory reproducibility of these assays is very good to excellent (31)(32)(33). Nevertheless, the comparability of HPV DNA quantitation results determined by different realtime PCR assays is unclear.…”