Women infected with HIV are at greater risk of anogenital human papillomavirus (HPV) infection and HPV-induced squamous intraepithelial lesions (SIL), as detected by cytology, or cervical intraepithelial neoplasia (CIN), as detected by histology [1–3]. HPV-16 is among the most common genotype detected in women without lesion whether infected or not with HIV, and accounts for half of cervical cancers and high-grade CIN (CIN 2,3) [4–6]. As most HIV-seropositive women are infected with HPV , biomarkers that could help identify women at greater risk of CIN progression would be desirable. Several studies have suggested that a high HPV-DNA viral load could be a candidate marker for cervical CIN in HIV-seronegative [8–10] and HIV-seropositive [11–14] women. Increased quantities of integrated HPV-16 DNA have also been reported in CIN 2,3 . HPV integration is considered to be a key event in the progression of cervical lesions to cancer [16,17]. HPV-16 integration was recently demonstrated in early stages of CIN [15,18,19].
The role of HPV-16 viral load and HPV-16 integration in the screening of cervical lesions remains unclear. HPV-16 integration has not been studied in HIV-seronegative women without CIN, and has not been evaluated in HIV-seropositive women with or without CIN. To define the association between HPV-16 viral load, integration and cervical disease, we measured episomal and integrated HPV-16 DNA with real-time polymerase chain reaction (PCR) assays on specimens collected in the course of a cohort study examining the natural history of HPV infection in HIV-seropositive and seronegative women. Our results suggest that HPV-16 DNA viral load is greater in women with cervical disease. Specimens from women without lesion, however, contained a mixture of integrated and episomal forms, impeding the potential usefulness of these real-time PCR tests to predict CIN progression.
Materials and methods
Study population and study design
Subjects were selected from women infected with HPV-16 participating in the Canadian Women's HIV Study between May 1993 and March 2002. The Canadian Women's HIV Study is a cross-sectional and cohort study that investigates determinants of HPV persistence in women infected or at risk of HIV [20–22]. As described elsewhere, 1055 women were enrolled from sexually transmitted disease, primary care clinics, and outpatient HIV care clinics across Canada. Women were eligible if they provided written informed consent, were seropositive for HIV, or were seronegative for HIV but at risk of sexually transmitted diseases [20,22]. A standardized questionnaire was administered upon study entry and at 6-month intervals for all participants. At each visit, a vaginal tampon specimen was obtained as previously described . A Pap smear was then obtained by the use of a cytobrush and Ayres spatula, and cervicovaginal lavage (CVL) was performed with 10 ml sterile phosphate-buffered saline sprayed on the ectocervix with a syringe. Cell suspensions were lysed with 0.8% Tween and digested with proteinase K . An aliquot of 5 μl from each processed sample was amplified for β-globin DNA with PC04-GH20 . β-Globin-positive samples were tested for HPV DNA detection and typing using the MY09–MY11–HMB01 consensus L1 PCR and type-specific probes [21,23].
For all HIV-seropositive women, CD4 cell counts, Pap smears, vaginal tampons and CVL were obtained upon study entry and at 6-month intervals [20,21]. For HIV-seronegative women, vaginal tampons were obtained at inclusion and at 6-month intervals, whereas CVL and Pap smears were collected at one-year intervals. Cytology smears were interpreted in one central pathology laboratory and confirmed by one pathologist using the 1991 Bethesda classification . Colposcopy was performed systematically to participants with high-grade squamous intraepithelial lesions (HSIL) on cytology smears and was suggested to participants with smears showing low-grade squamous intraepithelial lesions (LSIL). Colposcopy and biopsy results were made available to the study investigators. Of the 732 HIV-seropositive and 323 HIV-seronegative women screened for cervical HPV infection at their initial study visits, 366 HPV-infected women (207 HIV-seropositive with a mean of follow-up of 27.3 months, 159 HIV-seronegative with a mean follow-up of 17.9 months) were followed prospectively.
Overall, 132 (12.5%) of 1055 women had at least one CVL containing HPV-16 DNA at baseline or during follow-up visits. For the current publication, data were limited to 75 women (58 HIV seropositive, 17 HIV seronegative): 39 had colposcopy, 24 had over a period of at least 12 months three consecutive normal smears, seven had two or more consecutive smears with LSIL, two had one smear with HSIL, and three were infected by an African or Asian variant. None of the 57 HPV-16-positive women excluded from the study had had colposcopy, 26 had completed one visit only, two had completed two visits only, 12 provided normal smears but at fewer than three visits, nine did not provide Pap smears, six were HPV-16 positive at the last visit only, and two had large intervals between visits greater than 1.5 years.
Real-time polymerase chain reaction for HPV-16 viral load and integration
HPV-16-positive CVL from 75 women were further screened for the presence of inhibitors by real-time PCR. Internal controls for HPV-16 E6 and E2 were synthesized by amplifying the pSP65 plasmid with primers containing pSP65 sequences (20 underlined bases) to which HPV-16 sequences were appended at the 5′ end: ci-16-E6-F [5′–GAGAACTGCAATGTTTCAGGACC
–3′, underlined sequences are base pairs (bp) 1–20 from pSP65] and ci-16-E6-R (5′–ATAGTTGTTTGCAGCTCTGTGC
–3′ bp 407–426 of pSP65) for the E6 gene and ci-16-E2-F (5′–AACGAAGTATCCTCTCCTGAAATTATTAG
–3′ and ci-16-E2-R (5′–CCAAGGCGACGGCTTTG
–3′) [25,26]. Amplification of pSP65 with these primers generated pSP65 fragments flanked at both ends by primer sequences used for HPV-16 amplification. One thousand copies of HPV-16 E6, HPV-16 E2 and β-globin internal controls were mixed in separate capillaries with 2 μl CVL lysate and tested in a Light Cycler PCR and detection system (Roche Molecular Systems, haval, Quebec, Canada) with primer pairs described below, as previously published for E6 and β-globin [25,26]. The presence of PCR inhibitors was suspected when the 1000 copy internal control generated a signal corresponding to less than 700 copies for at least one internal control [25,26]. The latter cut-off was established in previous work considering the variability of the assays and the demonstration of inhibition by dilution of lysates . Forty-six samples containing inhibitors [inhibition of both internal controls (n = 4), HPV-16 internal control inhibition only (n = 34) and β-globin internal control inhibition only (n = 8)] were retested with internal controls after dilution of lysate (n = 38) or after DNA purification with Master Pure (n = 8).
Two microlitres of lysate or processed sample without inhibition were then tested in duplicate in separate capillaries for quantitation of HPV-16 E6 and E2 genes, and β-globin DNA using hydrolysis probes as described previously . The 16-E6-PRO probe and primers 16-E6-F and 16-E6-R were utilized for HPV-16 E6 quantitation, whereas HPV-16 E2 gene was quantitated using probe 16-E2-PRO and primers 16-E2-F and 16-E2-R . For both assays, the 20-μl reaction mixture contained 1× DNA Master Hybridization Probe Mix containing the Fast Start Taq DNA polymerase (Roche Molecular Biochemicals), 4 mM magnesium chloride, 0.3 μM of each HPV-16 primer, 50 nM of the fluorogenic probe and 2 μl of processed sample. For β-globin, each sample was amplified in duplicate with 50 nM of β-globin probe U62049, 0.3 μM of primers U61992 and L62240, 4.5 mM magnesium chloride, and 1× DNA Master Hybridization Probe [9,25,26]. Cycling parameters were as described previously [9,25,26].
Cycle thresholds were compared with those of an HPV-16 titration curve obtained by serial 10-fold dilutions of an HPV-16 plasmid, kindly provided by Professor zur Hausen, in a fixed amount of 75 ng human fibroblast DNA in 10 mM Tris-HCl (pH 8.2). Titration curves of human DNA were obtained by serial dilutions of a stock of human genomic DNA (Roche Molecular Biochemicals) in 10 mM Tris-HCl (pH 8.2). HPV-16 E6 and E2 loads were expressed as the number of HPV-16 copies per microgram of human DNA. The integrated HPV-16 viral load was calculated by subtracting the copy numbers of E2 (episomal) from the total copy numbers of E6 (integrated and episomal). Total, episomal and integrated HPV-16 loads were then log-transformed.
Molecular variant analysis by polymerase chain reaction sequencing
HPV-16 isolates were further characterized by PCR sequencing of a 364-bp segment within the long control region (LCR)  and of the complete E6 gene . HPV-16 amplicons were generated with 2.5 units of Expand High Fidelity PCR enzyme (Boehringer Mannheim, Laval, Quebec, Canada) and purified with the QIAquick gel extraction kit protocol (Qiagen Inc., Mississauga, Ontario, Canada). Direct double-stranded PCR-sequencing was performed with the fluorescent cycle-sequencing method (BigDye terminator ready reaction kit; Applied Biosystem, Foster City, California, USA) on an ABI Prism 3100 Genetic Analyser system. HPV-16 isolates were classified into two broad categories: prototype and non-prototype strains, and into one of the five lineages as described by Yamada et al. .
HPV-16 viral loads were measured in specimens collected before biopsy that could alter viral load measurements. Univariate analysis was first performed to identify factors significantly associated with SIL on cytology or with CIN on biopsy, using Fisher's exact test for categorical variables and the Mann–Whitney U test for continuous variables. Analyses of cytology results were conducted by considering the visit for each participant with the highest grade of SIL. Given previous reports on the potential role of the E6 G350T variation in disease , it was specifically evaluated in relation to CIN. The magnitude of the association between measures of integrated and episomal HPV-16-DNA load and grade of CIN or SIL was assessed by logistic regression controlling for age, race and a factored variable combining the CD4 cell count and HIV status (HIV negative, HIV positive with CD4 cell counts > 500, 200–500, and < 200 cells/μl). The correlation between the HPV-16 viral load and age or CD4 cell count was measured with the Spearman rank correlation coefficient (ρ). HPV-16 viral loads from isolates of different HPV-16 LCR lineages were compared with the Mann–Whitney U test. All statistical tests were two-sided with statistical significance set at P < 0.05.
The baseline characteristics of the 75 women studied are presented in Table 1. There was no correlation between the total HPV-16 load and age (R = −0.13, P = 0.28) or HPV-16 viral load and CD4 cell count (R = −0.20, P = 0.09). We could not correlate the HPV-16 viral load with the plasma HIV viral load, because several visits had been completed before the advent of HIV viral load testing. When compared two by two, there was a strong correlation between the total, episomal and integrated HPV-16 DNA copies measured in each sample (ρ values from 0.82 to 0.97; P < 0.0001 for each comparison). As shown in Table 2 and Fig. 1 for the 72 participants with cytology smear results, the total, episomal or integrated HPV-16 loads were significantly higher in women with LSIL or HSIL than in women with normal smears (P < 0.02 for each comparison). Similar results were obtained when the first HPV-16-positive visit for each participant was analysed (data not shown). The differences between HPV-16 loads in women with HSIL and LSIL did not reach statistical significance (P > 0.15). There were no differences in age, CD4 cell count, co-infection with types other than 16, HAART, race or HIV status, between women with normal smears, LSIL or HSIL (P > 0.10 for each comparison, data not shown). Controlling for age, race, HIV status and CD4 cell count, a greater quantity of total (OR 1.6, 95% CI 1.1–2.6; P = 0.02), episomal (OR 1.6, 95% CI 1.1–2.5; P = 0.02), and integrated (OR 1.1, 95% CI 1.1–2.0; P = 0.05) HPV-16 DNA was measured in women with HSIL than in women with normal cytology.
As cytology may misclassify cervical disease, we investigated the association between the HPV-16 viral load and cervical disease in 39 women who had undergone colposcopy and biopsy of suspicious lesions and in 24 women who had not undergone colposcopy but had had three consecutive normal smears. The quantities of total, episomal and integrated HPV-16 DNA (Table 3) in CVL from women with high-grade CIN (CIN 2,3) were at least two logs greater than in normal women (P < 0.002). Although CIN 2,3 contained at least 10 times more HPV-16 DNA copies than CIN 1, this difference did not reach statistical significance (P > 0.06). When only HIV-seropositive women were considered, similar results were obtained (Table 3). Southern blot analysis using a 32P-labelled HPV-16 probe was performed on DNA extracted from CVL from three women with a normal cervix and with a load of integrated HPV-16 above the detection threshold of Southern blot, and revealed the presence of integrated forms in all women (data not shown). In previous studies, coefficients of variations of real-time PCR assays for HPV quantitation reached at most 30% [25,26]. E6/E2 ratios greater than two were considered suggestive of integration. E6/E2 ratios greater than two were obtained for 21 out of 35 normal women (60%; including seven with ratios > 4), 11 out of 16 women with CIN 1 (69%) and seven out of 12 women with CIN 2,3 (58%). Excluding one women with CIN 3 who had no HPV-16 DNA detected, the range of HPV-16 integrated loads in five women with CIN 3 (104.20 to 108.46, median of 106.22) was similar to that measured in six women with CIN 2 lesions (0–108.30, median of 105.87). Three out of five women with CIN 3 and four out of six women with CIN 2 had E6/E2 ratios greater than two.
The CD4 cell count was significantly lower in women with CIN 2,3 than in normal women, whereas other factors were not significantly different (Table 4). No isolate carried the 131G variation previously associated with anal SIL . Controlling for HIV status, CD4 cell count and age, total (OR 3.5 95% CI 1.2–10.4; P = 0.02), episomal (OR 2.9 95% CI 1.2–7.4; P = 0.02) and integrated (OR 1.6 95% CI 1.1–2.6; P = 0.05) HPV-16 DNA loads remained significantly associated with CIN 2,3. Similar results were obtained with HIV-infected women only controlling for the CD4 cell count and age (data not shown).
We then evaluated whether HPV-16 polymorphism could influence HPV-16 viral load measures. The median HPV-16 DNA load in 57 women infected with European variants (median of 105.98, range of 103.67 to 108.88) was higher than in three women infected with Asian LCR variants (median of 103.32, range of 0–105.71; P = 0.04), but similar to nine women infected with African variants (median of 105.08, range of 0–108.48; P = 0.81). A variation at nucleotide 145 in the Asian variant was located in the middle of the probe. Samples with the latter variant were retested with an HPV-16 E6 assay that used primers and probe perfectly homologous with the E6 sequence of non-European variants [32,33]. The mean ratio of HPV-16 E6 load measured with the latter assay and the original assay reached 1.3 ± 0.5. This second HPV-16 E6 assay confirmed the lower total HPV-16 load obtained with the Asian variant (data not shown).
Finally, as reported by another group , the amount of cellular DNA collected per microlitre of processed CVL was greater for women with CIN 2,3 (median 0.052 μg, range 0.030–0.159; P = 0.007) but not CIN 1 (median 0.03 μg, range 0.012–0.063; P = 0.11) than in women with normal cervices (median 0.011 μg, range 0.003–0.026).
This study shows that a wide spectrum of episomal, integrated and total HPV-16 DNA loads can be measured with real-time PCR in a population of sexually active women. The measures of episomal and integrated HPV-16 DNA were obtained by a truly quantitative assay that not only normalized the amount of HPV-16 DNA against the quantity of host DNA collected, but also screened for the presence of PCR inhibitors. Our work clearly shows that these two adjustments are mandatory when cell lysates are analysed. As reported previously by two groups [9,34], the cellular content of genital specimens was greater in women with CIN 2,3. HPV-transformed cells may express fewer intercellular adhesion molecules and may be sampled more readily . Several samples contained inhibitors that would have altered our evaluation of HPV-16 or cellular DNA copies. The prospective design of the Canadian Women's HIV study also permitted an analysis of HPV-16 loads considering the highest grade of SIL obtained on consecutive visits. However, our study has limitations. Most women recruited were infected with HIV or were at risk of HIV infection. The small number of participants with lesions did not allow us to test possible associations of age or HPV-16 polymorphism with CIN 2,3, although we did find significant associations between CD4 cells and HPV-16 viral loads with CIN 2,3.
Initial studies using insensitive tools detected integrated forms only in high-grade lesions or cervical cancer [17,35]. As demonstrated here, the presence of integrated forms is difficult to quantitate because of the frequent occurrence of episomal and integrated forms in the same sample [15,36]. More sensitive technologies have demonstrated the presence of integration in precancerous disease [15,18,19,37]. One study demonstrated that nearly half of 92 samples collected from young women with LSIL contained integrated HPV-16 , whereas similar results were obtained by another group in another population . Another group reported that nearly all CIN lesions contained integrated forms . Our work is the first extensive report demonstrating integration in normal women. Mixed forms of episomal and integrated HPV-16 can be detected early during infection, before the presence of HPV-induced lesions. Possibly, E2 could still be disrupted without integration, but this possibility has not yet been reported. Some integrated HPV-16 forms may have been missed because only the most frequently disrupted region of E2 was analysed.
The measure of integration with real-time PCR is a novel approach. Differences between E6 and E2 viral loads allowing the measurement of HPV-16 integration may reflect technical limitations of the assays. Real-time PCR assays for HPV quantitation have very good intrarun and interrun reproducibility, with coefficients of variation below 30% [25,38,39]. Similar HPV-16 viral load values are obtained when samples are diluted several fold . In the current study, E6 and E2 were quantitated in duplicate and coefficients of variation were less than 18%, mostly under 10% (data not shown). A high level of concordance was found between HPV-16 loads estimated with HPV-16 E6 and L1 PCR assays (submitted). The slopes of HPV-16 DNA titration curves with the HPV-16 E6 and E2 PCR assays were similar (data not shown), suggesting similar amplification efficiency with the HPV-16 prototype. It is thus unlikely that selective inhibitors to E2 explain the lower HPV-16 E2 viral load results because no inhibition was detected with the internal control for E2 only.
More studies are needed to obtain a better understanding of the meaning of detecting integrated forms in normal women. Although the performance of the E6 and E2 assays were similar, an analysis of the E2 polymorphism to ensure that polymorphic sites at primer or probe binding sites are not responsible for the differences between the E6 and E2 assays would help define the usefulness of these assays. Although the amplification efficiency of the three PCR assays was similar in vitro, they could differ in complex nucleic acid environments.
To date, conflicting results have been obtained from various studies on the association between high HPV viral loads and cervical SIL or CIN [8–13,32,34,40–52]. Although cervical carcinoma in situ has been found in women with consistently high HPV-16 viral loads, HSIL may contain low HPV-16 loads [8,9,45]. Several groups have also demonstrated that HIV-seropositive women with HSIL were infected with greater quantities of HPV DNA [11–14]. We have shown here that high HPV-16 loads are associated with HSIL or CIN 2,3. As reported by others, the important overlap of HPV-16 viral load values between normal women and those with lesions could limit the usefulness of viral load measurements and could partly explain the inconsistencies between studies [12,14]. Moreover, the presence of CIN 1 surrounding CIN 3 lesions can alter HPV viral load estimations and restrict the clinical usefulness of this marker . Eight studies, including our work, have studied the HPV-DNA viral load in exfoliated cervical cells from HIV-infected women [11–14,44,54,55]. Higher HPV viral loads have been measured by some in women with lower CD4 cell counts [12,14]. The lack of correlation between the HPV-16 load and CD4 cells reported here is in line with a recent report  demonstrating that prevalent and incident HPV-16 infections were more weakly associated with the immune status than with other HPV types, suggesting that HPV-16 may better avoid immune surveillance. Asian variants were detected at lower viral loads than European isolates, a finding that did not reach statistical significance in a previous report from another cohort .
Our findings demonstrate that women who are infected by higher HPV-16 viral loads are more likely to have significant cervical lesions. However, the important overlap between disease grades of HPV-16 viral load does not permit a clear classification of participants. Prospective studies involving a greater number of HPV-16-infected women are needed to define the predictive value of the integrated HPV-16 load versus the total or episomal load for progression. Prospective studies on types other than 16 could also establish whether the same association could be found across high-risk types. No additional information was gained in our study by measuring integrated or episomal over total HPV-16 DNA loads.
The authors would like to thank Mme Diane Gaudreault and Mme Diane Bronsard for processing genital samples. They would also like to thank Fabrice Rouah for maintaining the database.
Conflict of interest disclosure: the authors do not have commercial or other associations that might pose a conflict of interest.
Sponsorship: This study was supported by the Canadian Institutes for Health Research and by le Réseau FRSQ Maladies Infectieuses SIDA. The Canadian Institutes for Health Research supports the Canadian Women's HIV Study cohort. F.C. is a national researcher supported by the Fonds de la Recherche en Santé du Québec (FRSQ).
The Canadian Women's HIV Study Group includes the following investigators. Halifax: Janet Conners, Rob Grimshaw, David Haase, Lynn Johnston, Wally Schlech, Arlo Yuzicappi-Fayant. Hamilton: Stephen Landis, Fiona Smaill. London: Tom Austin, Ole Hammerberg, Ted Ralph. Montréal: François Coutlée, Julian Falutz, Alex Ferenczy, Catherine Hankins, Marina Klein, Louise Labrecque, Normand Lapointe, Richard Lalonde, John Macleod, Grégoire Noël, Chantal Perron, Jean-Pierre Routy, Emil Toma. Ottawa: Claire Touchie, Garry Victor. Québec: Louise Coté, Hélène Senay, Sylvie Trottier. Saskatoon: Kurt Williams. Sherbrooke: Alain Piché. Sudbury: Roger Sandre. Toronto: Louise Binder, Donna Keystone, Anne Phillips, Anita Rachlis, Irving Salit, Cheryl Wagner, Sharon Walmsley. Vancouver: Paula Braitstein, David Burdge, Marianne Harris, Deborah Money, Julio Montaner.
All participants provided written informed consent to participate. Ethics committees of each participating institution approved the Canadian Women's HIV Study protocol.
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