All clinical samples in this study were organized using the Weifang Cervical Lesions Screening cohorts, Shandong, P.R. China. Detection of the 21 HPV genotypes was performed by HPV GenoArray Test Kit (HybriBio Ltd. China) before enrollment of patients. 605 women who needed colposcopic evaluation for abnormal cervical screening in Affiliated Hospital of Weifang Medical University, China, between November 2017 and September 2018 were enrolled. Inclusion criteria are as follows: 1) Agreement of participation; 2) HR-HPV positive; 3) Indication for colposcopy ; 4) No hysterectomy; 5) No pregnancy. Women with cervical lesions not completely visible (unsatisfactory colposcopy) were excluded (Figure 1). In total, 273 women were eligible in the study. All participants signed an informed consent document before enrolment. Women’s age ranged from 21 to 63 years (38.4±9.0 years). Based on the number of HPV types in primary screening, women were divided into the following two groups: multiple HPV types (at least two or more genotypes of HR-HPV infection) and single HPV type (Only one genotype of HR-HPV infection).
At first, one physician (T. T. Wang) who was trained used cytobrush (Digene Cervical Sampler, USA) to collect exfoliated cells of endocervix (an area, which can be covered by cytobrush, bounded below by the ectocervix within a 0.5 cm radius of external orifice of uterus). Then, a different cervical brush (Thinprep ® Cytologic Test Sampler, USA) was used to collect exfoliated cells of ectocervix (an area, cannot be covered by cytobrush, bounded above by ectocervix over a 0.5 cm radius of the external orifice of uterus). Both specimens were separately transferred to specimen preserving medium (formalin), stored at -4°C and sent to laboratory for viral load testing using the commercially available Hybrid Capture II system (Digene, USA). Based on the anatomical sites of sampling, sampling methods were stratified into two groups: Simple endocervical sampling group, combination of endo- and ectocervical sampling group.
A standard colposcopic examination was performed by the same professional colposcopist (Y.Z. Liu). Cervical lesions showing aceto-white epithelium and/or atypical vessel changes (mosaic, punctation and tumour vessels) were measured under 15X magnification of colposcopy. Area (cm2) and location (the distance between external orifice of uterus and the below bound of lesions, cm) of lesions was quantified using colposcopy image processor. Based on the location of lesions, women were stratified into two groups: involvement of the area bounded below by the ectocervix within a 0.5 cm radius of the external orifice of uterus（≤0.5）, involvement of the area bounded below by ectocervix over a 0.5 cm radius of the external orifice of uterus (>0.5). For pathology diagnosis, colposcopy with a directed biopsy was performed as indicated and biopsy was evaluated by two pathologists who were blind to colposcopy or clinical information. All cases with disagreement were reanalyzed by another experienced pathologist. Based on colposcopy and pathology, the patients were stratified into three groups: Negative (negative for intraepithelial lesion or malignancy), LSIL (low-grade squamous intraepithelial lesion), HSIL/CC (high-grade squamous intraepithelial lesion and / or cervical cancer).
Detection of HPV–DNA
HPV genotyping by HybriMax was performed using an HPV GenoArray Test Kit (HybriBio Ltd., China). This assay can determine 21 HPV types, including 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), ﬁve low-risk HPV types (6, 11, 42, 43, and 44), and two unknown-risk types (53 and CP8304), by the ﬂow-through hybridization technique using HPV DNA ampliﬁed by PCR as described before .
Hybrid Capture II assay (Digene, USA) was performed on the cervical scrapings collected from endo- and ectocervix (bounded by the 0.5 cm radius of external orifice of uterus) separately using two different cervical brushes. Light measurements were quantified using a luminometer and were expressed by comparing the relative light units (RLU) of clinical samples with a positive control (PC). RLU/PC ratios (RLUs) were calculated as the ratio of the specimen luminescence to the luminescence of the 1.0 pg/mL HPV cut-off standard, which is known to represent a semi-quantitative value for the cumulative viral burden of one or more of the 13 oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). An RLU/PC ratio of 1 or higher was considered a positive result as proposed by the manufacturer (equivalent to 1 pg/mL of HPV–DNA).
All statistical analyses were made by means of the SPSS version 22.0 for Windows. Means, medians, ranges, standard deviations, 95% confidence intervals, standard errors of means were used as descriptive statistics. Viral load was measured as RLU/PC ratios (RLUs) and agrees with previous speciﬁcations in the hybrid capture assay. Viral quantiﬁcation data in RLUs were initially continuous measurements. RLUs were transformed into their logarithm (Log10). An index of Spearman correlation coefficient (rs) was applied to measure the association between viral load and variables. Multiple linear regression analysis was applied to relate the viral load with variables. The variables included in the model were age, histological severity, number of HPV types, area and location of the cervical lesions. A paired t-test was used to compare viral load with different sampling methods. Two-tailed P values less than 0.05 were considered statistically significant.