Validation of In-house HPV-16 DNA Quantitative Real-time PCR Testing
Keywords:
clinical sensitivity and specificity, HPV-16 DNA, in-house quantitative real-time PCR, Thai women, validationAbstract
Introduction: HPV is a causative agent of cervical cancer, and is the second leading cancer for Thai women after breast cancer. HPV-16 is the most common subtype associated cancer. Since cytology screening has become a problem that has a large number of borderline results, our purpose is to clinically validate the sensitivity and specificity of the in-house HPV-16 DNA quantitative real-time PCR assay test compared to a reference method, and validate its reliability in order to use it as a combined test for managing abnormal Pap smear samples and to determine if viral load is associated with the virulence of disease. Sixty five out-patients with borderline Pap smear results, from the ‘Gynecological Ward’ at Bhumipol Adulyadej hospital, Bangkok, Thailand, and 119 normal women presenting annual Pap smear screening were recruited for the study, between 2010 and 2012. All were determined by a colposcopy-directed biopsy as a reference method. The cervical scrape samples were taken to test for HPV DNA using a quantitative real-time PCR primer and specific probe for the L1 region of HPV DNA. The test was validated for clinical sensitivity and specificity, for the detection of HPV2 DNA in precancerous lesions (≥CIN2), and for its reliability. Out of 65 samples with borderline results, 45 cases were HPV-16 DNA positive (69.2%). Only 14/65 cases had precancerous lesions (≥CIN2). For clinical sensitivity, out of 14 of those cases, 12 patients were HPV-16 DNA detected (86%). For the clinical specificity of the test, in normal women; out of 96 normal women who were negative for colposcopy (without CIN2), 76 cases tested negative for HPV-16 DNA (80%). The viral load did not correlate to the virulence of disease. This test might be useful for health care practices as an adjunct to cytology follow–ups for women with borderline results.
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