BackgroundCervical lesions caused by integrated human papillomavirus (HPV) infection are highly dangerous because they can quickly develop into invasive cancers. However, clinicians are currently hampered by the lack of a quick, convenient and precise technique to detect integrated/mixed infections of various genotypes of HPVs in the cervix. This study aimed to develop a practical tool to determine the physical status of different HPVs and evaluate its clinical significance.MethodsThe target population comprised 1162 women with an HPV infection history of¿>¿six months and an abnormal cervical cytological finding. The multiple E1-L1/E6E7 ratio analysis, a novel technique, was developed based on determining the ratios of E1/E6E7, E2/E6E7, E4E5/E6E7, L2/E6E7 and L1/E6E7 within the viral genome. Any imbalanced ratios indicate integration. Its diagnostic and predictive performances were compared with those of E2/E6E7 ratio analysis. The detection accuracy of both techniques was evaluated using the gold-standard technique ¿detection of integrated papillomavirus sequences¿ (DIPS). To realize a multigenotypic detection goal, a primer and probe library was established.ResultsThe integration rate of a particular genotype of HPV was correlated with its tumorigenic potential and women with higher lesion grades often carried lower viral loads. The E1-L1/E6E7 ratio analysis achieved 92.7% sensitivity and 99.0% specificity in detecting HPV integration, while the E2/E6E7 ratio analysis showed a much lower sensitivity (75.6%) and a similar specificity (99.3%). Interference due to episomal copies was observed in both techniques, leading to false-negative results. However, some positive results of E1-L1/E6E7 ratio analysis were missed by DIPS due to its stochastic detection nature. The E1-L1/E6E7 ratio analysis is more efficient than E2/E6E7 ratio analysis and DIPS in predicting precancerous/cancerous lesions, in which both positive predictive values (36.7%-82.3%) and negative predictive values (75.9%-100%) were highest (based on the results of three rounds of biopsies).ConclusionsThe multiple E1-L1/E6E7 ratio analysis is more sensitive and predictive than E2/E6E7 ratio analysis as a triage test for detecting HPV integration. It can effectively narrow the range of candidates for colposcopic examination and cervical biopsy, thereby lowering the expense of cervical cancer prevention.