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Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples.

ABSTRACT: The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

SUBMITTER: Ardhaoui M

PROVIDER: S-EPMC8357094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAccording to international guidelines, Human Papillomavirus (HPV) DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more (CIN2+) lesions. The study aimed to assess whether HPV Selfy (Ulisse BioMed - Trieste, Italy), a full-genotyping HPV DNA test that detects and differentiates 14 high-risk HPV (HR-HPV) types, meets the criteria for primary cervical cancer screening described in the international guidelines, on clinician-collected as well as on self-collected samples.MethodsFor each participant woman, consecutively referring to Azienda Sanitaria Universitaria Giuliano Isontina (Trieste, Italy) and CRO-National Cancer Institute (Aviano, Italy) for the cervical cancer screening program, the following samples were tested: (a) a clinician-collected cervical specimen, analyzed with the reference test (Hybrid Capture®2 test, HC2) and HPV Selfy; and (b) a self-collected vaginal sample, analyzed with HPV Selfy. Enrolled women were also asked to fulfill a questionnaire about self-sampling acceptability. As required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with its reference test.ResultsHPV Selfy clinical sensitivity and specificity resulted non-inferior to those of HC2. By analysis of a total of 889 cervical liquid-based cytology samples from a screening population, of which 98 were from women with CIN2+, HPV Selfy showed relative sensitivity and specificity for CIN2+ of 0.98 and 1.00 respectively (non-inferiority score test: P = 0.01747 and P = 0.00414, respectively); the test reached adequate intra- and inter-laboratory reproducibility. Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen (0.92 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population.ConclusionsHPV Selfy fulfills all the requirements of the international Meijer's guidelines and has been clinically validated for primary cervical cancer screening purposes. Moreover, HPV Selfy has also been validated for self-sampling according to VALHUDES guidelines. Therefore, at date, HPV Selfy is the only full-genotyping test validated both for screening purposes and for self-sampling. Trial registration ASUGI Trieste n. 16008/2018; CRO Aviano n.17149/2018.

Project description:Purpose: Offering self-sampling of cervico-vaginal material for high-risk human papillomavirus (hrHPV) testing is an effective method to increase the coverage in cervical screening programs. Molecular triage directly on hrHPV-positive self-samples for colposcopy referral opens the way to full molecular cervical screening. Here, we set out to identify a DNA methylation classifier for detection of cervical precancer (CIN3) and cancer, applicable to lavage and brush self-samples.Experimental Design: We determined genome-wide DNA methylation profiles of 72 hrHPV-positive self-samples, using the Infinium Methylation 450K Array. The selected DNA methylation markers were evaluated by multiplex quantitative methylation-specific PCR (qMSP) in both hrHPV-positive lavage (n = 245) and brush (n = 246) self-samples from screening cohorts. Subsequently, logistic regression analysis was performed to build a DNA methylation classifier for CIN3 detection applicable to self-samples of both devices. For validation, an independent set of hrHPV-positive lavage (n = 199) and brush (n = 287) self-samples was analyzed.Results: Genome-wide DNA methylation profiling revealed 12 DNA methylation markers for CIN3 detection. Multiplex qMSP analysis of these markers in large series of lavage and brush self-samples yielded a 3-gene methylation classifier (ASCL1, LHX8, and ST6GALNAC5). This classifier showed a very good clinical performance for CIN3 detection in both lavage (AUC = 0.88; sensitivity = 74%; specificity = 79%) and brush (AUC = 0.90; sensitivity = 88%; specificity = 81%) self-samples in the validation set. Importantly, all self-samples from women with cervical cancer scored DNA methylation-positive.Conclusions: By genome-wide DNA methylation profiling on self-samples, we identified a highly effective 3-gene methylation classifier for direct triage on hrHPV-positive self-samples, which is superior to currently available methods. Clin Cancer Res; 24(14); 3456-64. ©2018 AACR.

Project description:BackgroundWHO has recommended HPV testing for cervical screening where it is practical and affordable. If used, it is important to both clarify and implement the clinical management of positive results. We estimated the performance in Lusaka, Zambia of a novel screening/triage approach combining HPV typing with visual assessment assisted by a deep-learning approach called automated visual evaluation (AVE).MethodsIn this well-established cervical cancer screening program nested inside public sector primary care health facilities, experienced nurses examined women with high-quality digital cameras; the magnified illuminated images permit inspection of the surface morphology of the cervix and expert telemedicine quality assurance. Emphasizing sensitive criteria to avoid missing precancer/cancer, ~ 25% of women screen positive, reflecting partly the high HIV prevalence. Visual screen-positive women are treated in the same visit by trained nurses using either ablation (~ 60%) or LLETZ excision, or referred for LLETZ or more extensive surgery as needed. We added research elements (which did not influence clinical care) including collection of HPV specimens for testing and typing with BD Onclarity™ with a five channel output (HPV16, HPV18/45, HPV31/33/52/58, HPV35/39/51/56/59/66/68, human DNA control), and collection of triplicate cervical images with a Samsung Galaxy J8 smartphone camera™ that were analyzed using AVE, an AI-based algorithm pre-trained on a large NCI cervical image archive. The four HPV groups and three AVE classes were crossed to create a 12-level risk scale, ranking participants in order of predicted risk of precancer. We evaluated the risk scale and assessed how well it predicted the observed diagnosis of precancer/cancer.ResultsHPV type, AVE classification, and the 12-level risk scale all were strongly associated with degree of histologic outcome. The AVE classification showed good reproducibility between replicates, and added finer predictive accuracy to each HPV type group. Women living with HIV had higher prevalence of precancer/cancer; the HPV-AVE risk categories strongly predicted diagnostic findings in these women as well.ConclusionsThese results support the theoretical efficacy of HPV-AVE-based risk estimation for cervical screening. If HPV testing can be made affordable, cost-effective and point of care, this risk-based approach could be one management option for HPV-positive women.

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Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples.

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