Peter C. Angeletti
Date of this Version
Kapie, KS (2018). Evaluation of a Human papillomavirus genotyping assay for cervical cancer screening in Tanzania. MS Thesis, University of Nebraska-Lincoln.
Abstract: Mucosal high-risk Human papillomavirus (HR-HPV) has been identified as the primary factor in causing cervical cancer, the most common cancer in women of low and middle-income countries (LMICs). Cervical cancer screening, incorporating the HPV test shown to be more advantageous than screening by visual inspection with acetic acid (VIA) alone. However, due to resources constrain these countries could not afford the available HPV tests. We, therefore, validated a cost-effective Multiplex PCR HPV (mPCR) assay, a recently developed assay for detecting sixteen genital HPV, fourteen HR-HPV and 2 low-risk genotypes. We postulate that this HPV multiplex assay will be at least as accurate as commercially available genotyping methods to detect HPV types (16,18,30,31,33,35,39,45,51,52,56,58,59 and 66) and two low-risk (LR) type 6 and 11. The assay demonstrates high sensitivity and specificity in detecting single and multiple HPV infections. Compared to a World Health Organization (WHO) validated assay, mPCR had an almost perfect agreement in detection of the sixteen HPV genotypes, 98% (102 out of 103 per samples); coefficient of agreement (k) value of 0.955 and 96.5% (110 out of 114 detection events); k value of 0.923. This assay may help in assessing cervical cancer in Tanzania and other LMICs when used together with Pap smear. In the second part of the study, we evaluated a mPCR melt-off assay for detection of the sixteen HPV mPCR products. We performed a two-step experiment; PCR amplification followed by a Real-time PCR melt-off assay using SYTO 82 Orange fluorescence dye in instead of Syber Green. We postulated that the assay could detect mPCR amplicons and would distinguish a few different genotypes of HPV, in a single reaction, if the amplicons had unique melting profiles. The assay was able to detect each of the sixteen HPV amplicons by their unique melting temperatures (Tms). When tested in triplicate experiments, each HPV amplicon gave consistent Tm measurements. However, we discovered that the assay could not clearly distinguish Tm peaks of more than 3 amplicons in a single sample. Instead, Tm peaks were merged together when there was less than a 1oC temperature difference of the Tms of the amplicons. Clear Tm peaks were observed when the Tms of the respective amplicon Tms were at least 2oC apart. The assay could still be simplified to detect one or two of the most abundant HR-HPV genotypes found in Tanzania. We predict that this assay would be cheaper than any commercial DNA test and could be used to triage women with lesions by helping to predict their risk for developing cervical cancer hence improve management of these women.
Advisor: Peter C. Angeletti