Persistent high-risk human papillomavirus (HR-HPV) infection is the main cause of precancerous and cancerous lesions of the cervix [4-5]. Viral load reflects the number of infected cells and viral copies in individual cells. High viral load has been suggested as a marker of nontransient infection . Therefore, a high viral load may also be associated with the progression of cervical lesions. However, the association between HPV viral load and cervical lesion grade is controversial. There could be undetermined confounding factors, such as age, number of viral types, area and location of cervical lesions [5,12-14]. Few studies have incorporated these factors into the analysis, so the influence of confounding factors cannot be excluded. In the present study, we conducted a multiple linear regression analysis to evaluate factors associated with HR-HPV load. To analyse the influence of sampling factors on viral load, two different sampling methods were used to obtain cervical specimens for viral load detection. The aim of this study was to explore the possible influencing factors of viral load.
In this study, the results showed that age was an independent factor for HR-HPV load and that a significantly increasing age correlated with a higher viral load. This result agrees with previous population-based studies [17-18], which showed that HR-HPV load was higher in older age. Different from previous studies, this study incorporated different variables into the analysis to adjust the effect of confounding factors and showed that age was an independent factor for HR-HPV load. However, contrary to our finding, Flores R et al. reported that HR-HPV load declined with age and was signiﬁcantly higher in younger women . They divided women into three age groups (15-24, 25–32, 32–79 years) and found that younger women presented higher viral load. This observation is understandable, and the effect of age on viral load is probably related to the balance between new acquisition of HR-HPV infections and viral clearance. Young women (<25 years old) may represent new exposures to HPV due to sexual debut when an immune response to HPV has not yet been established. Therefore, the rate of new acquisition of HR-HPV infections exceeds the clearance rate , favouring the rapid accumulation of infections, which might contribute to a high viral load at an early age. Older women (>45 years old) are more likely to experience physiological and immunological disorders during the menopausal transition. Such physiologic and immunologic dysregulation can result in an inability to establish an immune response to HPV, and a high level of HPV infections cannot achieve spontaneous clearance [13,21].
Several studies have reported the association of high viral load with the risk for cervical cancer and its precursors. A large number of studies used the Hybrid Capture II assay to measure viral load, and while some found viral load to be positively associated with increased severity of cervical lesions [6-7], others did not . Different from previous studies, the present study explored the relationship between HR-HPV load and cervical lesion severity when other cofactors were taken into consideration. In agreement with some investigators [6-7], this study showed a positive association between cervical lesion severity and viral load. However, this association lost significance when other cofactors, including age, presence of multiple HR-HPV infections and area of cervical lesions, were considered in multivariate analysis. This fact could be explained by the bias of Hybrid Capture II viral load. One major bias is that this test does not provide an evaluation of cell numbers, which vary substantially with different areas of cervical lesions. Moreover, high viral load detected by Hybrid Capture II may represent single or multiple HPV types among the 13 high-risk types detected by the kit, but this test does not provide the evaluation of the number of HR-HPV types. Therefore, these factors (area and presence of multiple HR-HPV infections) may confound the association between HR-HPV load and cervical lesion severity. Therefore, HR-HPV load determined by Hybrid Capture II alone may not be used as a molecular biomarker of risk for developing cervical (pre-) cancerous lesions.
The prevalence rate of concurrent multiple HPV genotype infections was reported to be approximately 20%-25% in several large studies [22-23], which was nearly consistent with our result (26.7%). The role of multiple infections in carcinogenesis, with synergistic or antagonistic effects, remains to be determined [24-25], although some studies have considered them to be a risk factor for HPV persistence and for preinvasive and invasive cervical lesions . In this study, we analysed the correlation between multiple HPV genotype infections and HR-HPV load, which was calculated by two sampling methods, when possible confounding factors were incorporated into the analysis. The data indicated that multiple HPV genotype infections presented with significantly higher viral loads than single HPV genotype infections, but not significantly so in the simple endocervical sampling method, probably due to the impact of sampling variation on viral load and the relatively small-scale population. This finding indicates that cervical lesions induced by multiple HPV genotype infections are more likely to be associated with increased viral copies.
Current data showed that a distinct upward trend of viral load paralleled the increasing area of cervical lesions in both univariate and multivariate analyses. This result corroborates previous studies that have reported that more severe lesions tended to be larger and to harbour more HPV viral copies [26-28]. Different from previous studies, this study incorporated different variables into the analysis to adjust for the effects of confounding factors and showed that the area of cervical lesions is an independent factor determining viral load. This finding indicates that viral load is strongly affected by the number of infected cells, which increases in parallel with the increasing area of squamous intraepithelial lesions. However, to explore the association between HPV viral load and cervical lesion grade, some studies normalized viral load by using human cells to adjust the absolute viral load (e.g., expressed by copies/10,000 cells) to lessen the impact of the variation of sample volume . This may lead to ignoring the effect of the number of infected cells on viral load.
HPV viral load estimated from cervical scrapings can be easily affected by sampling . The radius of the cytobrush, such as a Digene cervical sampler, which is commonly used in HPV tests, is 0.5 cm. It is limited to collecting specimens in the area of the external orifice of the uterus within a 0.5-cm radius. When cervical lesions extend to the area of the external orifice of the uterus over a 0.5-cm radius, the brush cannot fully reach them, resulting in the collection of an inadequate number of cells that would underestimate the actual viral load. Therefore, to evaluate the impact of sampling methods on viral load, we compared the currently used simple endocervical sampling method with a new method of a combination of endo- and ectocervical sampling. We found that the viral load with a combination of endo- and ectocervical sampling was significantly higher than that with simple endocervical sampling. However, there was no significant difference in viral load between the lesions located in the area of the external orifice of the uterus within and over a 0.5-cm radius. This may be explained by a possible biased selection of participants in our study, in that only a minor proportion of the enrolled women with cervical lesions involved the area of the external orifice of the uterus over a 0.5-cm radius.
To our knowledge, our study is the first to demonstrate that age and area of cervical lesions are independent factors determining viral load, which may contribute valuable data for future discussions related to the potential application of viral load measurement. Nevertheless, several limitations also need to be addressed. First, the number of women with cervical lesions involving the area of the external orifice of the uterus over a 0.5-cm radius is too limited to draw a definitive conclusion for the effect of location of the cervical lesions on viral load and specimen collection methods. In addition, clinical data collected in one institution limit our statistical power to draw definitive conclusions for populations worldwide. Finally, the sample size in our study hinders our capacity to evaluate the association of HR-HPV load with other potential confounders, such as sampling physicians and individual HPV types. Further intensive clinical setup and laboratory investigations are therefore needed.