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).
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.
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