We measured HPV DNA load for four high-risk types (16, 18, 31, 45) with real-time PCR on a set of samples collected prospectively in young women. These four genotypes are amongst the most frequently detected in cervical cancer. Contrary to cross-sectional studies of older women, the four HPV genotypes were detected at similar loads and were not substantially different between women with single and multiple type infections [1, 11, 35].
The quantitative real-time PCR assays we utilized to estimate HPV loads were specific and reproducible [25, 28]. The number of HPV DNA copies was normalized for cell content by quantitation of β-globin DNA. The HPV-16 integration assay was devised considering the genetic diversity of HPV-16 [28, 36]. Using type-specific quantitative assays allowed isolating the effect of HPV type loads in multiple type infections. The cohort design also allowed testing longitudinal correlations between pairs of visits. Consistent measurements for HPV types 16, 18, and 31 were shown for the five visits with evidence of correlation between loads among visits within subjects, whenever statistical precision was sufficiently high.
As reported by others, younger woman (<25 years) harbored higher HPV loads than those >25 years of age . These women were possibly exposed to HPV while they were immunologically naïve to HPV. We also observed a somewhat greater HPV burden among current and former smokers, although not consistently for all four HPV types. This finding could be explained by a possible defective cell-mediated immunity against HPV induced by tobacco . Results from this cohort as well as those of others suggest that tobacco smoking may increase the duration of high-risk HPV infection [23, 38]. This could be explained in part by the increased replication of HPV in women exposed to tobacco.
Although the regular use of condoms protects against most sexually transmitted infections, they are not as efficient against HPV infection . We found a trend with the consistent use of condoms for having higher HPV loads for most genotypes. These results could be biased because use of condoms may be associated with risky sexual behavior or exposure to new sexual partners . Condom use has been associated in one study with regression of CIN and clearance of HPV . Although oral contraceptive use did not modify the duration of high-risk HPV infection in our cohort , HPV-18 DNA loads were markedly increased in women on oral contraceptives. Oral contraceptives may also be a proxy for a higher number of sexual partners.
HPV-16 and -18 loads were good predictors of the duration of infection with these types but the same finding was not held for HPV-31 and -45. HPV-16 load has been reported by others to be a stronger predictor for persistence or lesions than HPV-18, 31 or 33 loads . There was a clear dose-response relationship between HPV load and persistence of HPV-16 and HPV-18 infections. We found that clearance rates depended largely on the level of HPV load. Viral-host interactions play an influential role in the clearance of viral infections . HPV has developed several mechanisms to evade the host immune system . Functional differences between HPV-encoded proteins could also explain why some types and variants have a better viral fitness with a greater ability to persist [14, 41, 43]. For HPVs 16 and 18, viral loads were greater with HPV co-infections at different visits (sequential) than concurrent co-infection. In recent studies, two or more oncogenic HPV types diagnosed concurrently did not confer an additional risk of developing lesions [44, 45]. All but one study confirmed that sustained or increased viral loads, especially with HPV-16, were predictive of persistent infection [3, 5, 8, 11]. In a cohort of nearly 6,000 women in France, women with HPV loads above 10 pg/ml were less likely to clear the infection, irrespective of the age of participants . Similarly, another cohort study conducted in the Netherlands reported that women with low HPV-16 loads were five times more likely to clear HPV-16 infection . In a third study conducted in Brazil, there was a dose-response relationship between increasing viral loads and risk of incident abnormal smear over time .
HPV-16 integration often results in the disruption of the E2 gene, leading to uncontrolled expression of HPV-16 oncoproteins [14, 17]. HPV-16 integration could thus occur in the course of persistent HPV-16 infection and increase the likelihood of progression to higher grade lesions. Integration of the HPV-16 genome was believed to occur at the CIN-2,3 stage and beyond . Recent studies support that integration can occur even in the absence of CIN [15, 28]. In one study conducted in women age 15 to 19 years old , disruption of the E2 gene was demonstrated in up to 25% of incident HPV-16 infections, suggesting that HPV-16 E2 disruption was a common event occurring early during infection. In contrast to this latter study, HPV-16 E2 disruption was rare in our study population, perhaps because unlike Collins et al  who studied the entire E2 gene for disruption our assay only focused on the hinge region of E2. While disruption of E2 is thought to occur more frequently in the very early phases of infection in younger women at least one other study of older women followed longitudinally, observed approximately 50% of women with persistent or transient infections to have mixed integrated and episomal forms . However, unlike our study, over 50% of these participants had Pap smear abnormalities. Furthermore, HPV integration was not found to be associated with persistence . Results from other cohorts are needed to assess the rate of integration and to clarify the role of HPV integration on persistence at early stages of infection.
There are some limitations to our study. We cannot exclude the possibility that we may have underestimated HPV-16 integration due to HPV-16 disruption during integration in areas outside of the E2 hinge region. Nevertheless, HPV-16 E2 hinge is the most frequently disrupted site in studies conducted in North America .
The analytical sensitivity of real-time PCR assays for quantitation of HPV-16 E6 and E2 DNA utilized in this study has been demonstrated previously and shown to be excellent [18, 28, 26]. These reagents were optimized to avoid any influences of viral polymorphism on the efficiency of HPV-16 DNA amplification . Nevertheless, the clinical sensitivity and specificity of these assays to detect HPV integration have not been thoroughly assessed. Reconstitution experiments have demonstrated that HPV-16 integration is detected with real time PCR assays measuring E6 and E2 when integrated HPV-16 forms are in 100-fold excess of episomal HPV-16 DNA . Theoretically, a HPV-16 E6/E2 ratio above 1.0 could suggest integration. However, previous work on HPV-6, HPV-16 and -33 have demonstrated that HPV E6/E2 ratios below 2.0 results from assay variability rather than true differences between E6 and E2 quantities [2, 18, 29, 34]. Real time PCR tests may thus be falsely negative for HPV-16 integration in the presence of low quantities of integrated forms and high quantities of episomal forms.
We could not confirm the presence of HPV-16 integration with a standard technique identifying the presence of HPV-human DNA junctions in the only sample with a HPV-16 E6/E2 ratio above 2.0. However, the quantity of sample that could be analyzed was limited. A recent report using a similar technique to demonstrate the presence of HPV-human junctions did not find HPV-16 integration in specimens from women with low-grade SIL . Real-time PCR assays may be helpful to detect integrated HPV forms but further studies on greater number of specimens comparing different techniques for detection of integrated HPV-16 need to be conducted.
In our study, few women had abnormal smears, reducing our power to test the associations between HPV load and lesion outcomes. The time interval between visits can influence the assessment of persistence and clearance. The majority of women in our cohort returned within 6 months of each visit and there was only a small proportion of women whose time interval between visits extended beyond one year . The association between higher viral loads and persistence would only be distorted if it had been associated differentially with time between visits. Given that the participants were unaware of their HPV and viral load status, this is an unlikely scenario. It is also unlikely that their behaviour and other risk factors will change in this short span of time. The same associations between HPV loads and persistence were obtained when HPV persistence was defined more stringently by using three consecutive HPV-positive visits for the same type.
Consecutive detection of HPV DNA is due to either ongoing viral replication, reactivation of latent infections or new infections [48, 49]. The design of our study cannot discriminate between these three possibilities. Participants considered as having persistent infection could have indeed, been reinfected with another isolate of the same HPV type. However, this seems unlikely because women with persistent infection were all infected with the same intratypic HPV variant. Both prevalent and incident HPV infections were included in our analysis of persistence, increasing the power of our analyses. Although it may not have a direct implication on HPV clearance, the exact duration of prevalent cases is unknown. By including them we could have introduced a bias because a greater proportion of prevalent HPV infections represent persistent infections compared to incident infections. However, our conclusions did not change when we restricted our analyses to incident infections (data not shown). We also analyzed the entire cohort to utilize the clustered binary outcome of persistence, by using the 'visit number' as panel variable in logistic GEE models. Apart from the gain in precision when using multiple visits from the same women, the findings from these models were in agreement with those from Cox models that analyzed clearance in individual women by tertile of viral load. Therefore, we conclude that the bias incurred due to inclusion of prevalent cases is minimal. We also doubt that misclassification of HPV status may have affected our results since there were very few women with persistent type-specific infections who had an intervening visit with a negative HPV test result and more than 80% of the same type persistent infections occurred during consecutive visits .