Objective: Our objective was to verify the sensitivity and specificity of dual immunocytochemistry staining for p16 and Ki-67 in liquid-based samples (the “dual” assay) for cervical lesion screening, compared to biopsy findings and human papillomavirus (HPV) DNA molecular detection. Study Design: Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values for the “dual immunocytochemistry assay” were calculated and compared to histopathological results and to high-risk HPV DNA detection in adult women or teenagers submitted to cervical cancer screening. Results: A total of 151 women were included. The majority (96.2%) of those with negative dual assay results had lower biopsy grades (p < 0.001). Women with cytology results suggestive of cervical cancer had positive dual immunocytochemistry assay results more frequently (p < 0.001), and these positive results were also significantly associated with biopsy findings (p < 0.001) and with high-risk genotype HPV infection (p = 0.007). Specificity and PPV for the dual assay were 0.972 (0.855–0.999) and 0.800 (0.284–0.995), respectively, and 1.000 (0.590–1.000) and 1.000 (0.631–1.000) for HPV detection. Conclusions: The dual immunocytochemistry assay had high specificity and PPV. It reveals a persistent HPV infection, avoiding the need for new tissue collections for biopsies or hybrid capture.

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