No abstract

Higher HPV-16 DNA loads are associated with cervical lesions detected by either histology or cytology. No additional information is gained by measuring integrated or episomal over total HPV-16 DNA loads.

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 (HPV-16) viral load in cervicovaginal lavage samples collected from 66 human immunodeficiency virus-seropositive women was inversely correlated with blood CD4 count (P ‫؍‬ 0.002). HPV-16 viral load was 81-fold higher in women with cervical smears suggestive of high-grade lesions (median, 4,425,883 copies/ g of DNA) than in women with normal smears (median, 54,576), controlling for age (P ‫؍‬ 0.006).Oncogenic human papillomaviruses (HPV) cause squamous intraepithelial lesions (SIL) and cancer of the uterine cervix in human immunodeficiency virus (HIV)-seronegative and -seropositive women (2). HIV-seropositive women are at increased risk compared to HIV-seronegative women for cervical HPV infection and SIL (9). Although HPV infects the genital tract of most HIV-seropositive women, only a minority of HPVinfected women develop persistent HPV infection that may progress into SIL and cancer (2). Biomarkers of HPV infection that could identify HPV-infected women at high risk for persistent infection and SIL would improve screening algorithms for management of HPV-induced cervical lesions.Several studies suggested that an increased HPV DNA viral load could be a candidate marker for the presence of cervical SIL in HIV-seropositive women (12,17,18). Similar studies with HIV-seronegative women on the predictive value of HPV viral load for high-grade-SIL (HSIL) detection yielded conflicting results (4,8,10,13,14,19). Some of the assays used in the latter studies were not HPV type specific or were semiquantitative, while assays using PCR did not take into account the presence of amplification inhibitors or did not estimate the quantity of cellular DNA tested. The latter two factors have been shown to influence HPV viral load determinations (7,14).A recent study validated a real-time PCR assay that measures the quantity of human cells and HPV DNA contained in biological fluids and uses internal controls to screen for the presence of PCR inhibitors (7). That study also reported that samples could selectively inhibit HPV-16 amplification (7). We present here cross-sectional results of cervical HPV-16 viral loads measured in a cohort of Canadian HIV-seropositive women. HPV-16 viral loads measured in cervicovaginal lavage (CVL) samples correlated with blood CD4 ϩ counts and were associated with cervical SIL.Women infected with HPV-16 were selected from participants recruited in The Canadian Women's HIV Study, a crosssectional and cohort study on the relationships between HPV and HIV infection and development of cervical SIL (3). Participants in the study were recruited from 1993 to 2000 across Canada from clinics involved in the care of HIV-infected patients and gave written informed consent (3). Ethics committees of each participating institution approved the study protocol. The demographic characteristics of the HIV-seropositive women have been described elsewhere (3). CVL samples from 732 HIV-seropositive women were screened at inclusion and at 6-month intervals for HPV infection using the MY09-MY1...