A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens
- 1 Downloads
Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin–labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.
KeywordsHigh-risk human papillomavirus (HR-HPV) FFPE cervical samples Cervical cancer Microplate hybridization Fluorometric assay
microplate colorimetric hybridization assay
microplate fluorometric hybridization assay
type-specific nested PCR
The authors wish to thank Mr. H. Argasi at the Research Consolation Center (RCC) at Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.
This work is based on a thesis in Virology by Negin Nikouyan (Project No. 93-7242), supported by Shiraz University of Medical Sciences, Shiraz, Iran.
Compliance with ethical standards
All the clinical specimens were obtained from the pathology department archive with local Ethics Committee approval at the Namazi Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. A statement of ethical approval is required to appear before the references for studies involving human or animal subjects.
Conflict of interest
The authors declare that they have no conflict of interest.
- 1.Plummer M, Schiffman M, Castle PE, Maucort-Boulch D, Wheeler CM (2007) A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion. J Infect Dis 195(11):1582–1589. https://doi.org/10.1086/516784 CrossRefPubMedGoogle Scholar
- 2.Rodriguez AC, Schiffman M, Herrero R, Hildesheim A, Bratti C, Sherman ME, Solomon D, Guillen D, Alfaro M, Morales J, Hutchinson M, Katki H, Cheung L, Wacholder S, Burk RD (2010) Longitudinal study of human papillomavirus persistence and cervical intraepithelial neoplasia grade 2/3: critical role of duration of infection. J Natl Cancer Inst 102(5):315–324. https://doi.org/10.1093/jnci/djq001 CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Jaisamrarn U, Castellsague X, Garland SM et al (2013) Natural history of progression of HPV infection to cervical lesion or clearance: analysis of the control arm of the large, randomised PATRICIA study. PLoS One 8(11):e79260. https://doi.org/10.1371/journal.pone.0079260 CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Mizushima T, Asai-Sato M, Akimoto K, Nagashima Y, Taguri M, Sasaki K, Nakaya MA, Asano R, Tokinaga A, Kiyono T, Hirahara F, Ohno S, Miyagi E (2016) Aberrant expression of the cell polarity regulator aPKClambda/iota is associated with disease progression in cervical intraepithelial neoplasia (CIN): a possible marker for predicting CIN prognosis. Int J Gynecol Pathol 35(2):106–117. https://doi.org/10.1097/pgp.0000000000000228 CrossRefPubMedGoogle Scholar
- 6.Mitra A, MacIntyre DA, Lee YS, et al. (2015) Cervical intraepithelial neoplasia disease progression is associated with increased vaginal microbiome diversity ;5:16865. doi: https://doi.org/10.1038/srep16865 https://www.nature.com/articles/srep16865#supplementary-information
- 9.Renshaw AA, Davey DD, Birdsong GG, Walsh M, Styer PE, Mody DR, Colgan TJ, College of American Pathologists Comparison Program in Cervicovaginal Cytology (2003) Precision in gynecologic cytologic interpretation: a study from the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Pathol Lab Med 127(11):1413–1420. https://doi.org/10.1043/1543-2165(2003)127<1413:pigcia>2.0.co;2 CrossRefPubMedGoogle Scholar
- 10.Castro FA, Koshiol J, Quint W, Wheeler CM, Gillison ML, Vaughan LM, Kleter B, van Doorn LJ, Chaturvedi AK, Hildesheim A, Schiffman M, Wang SS, Zuna RE, Walker JL, Dunn ST, Wentzensen N (2015) Detection of HPV DNA in paraffin-embedded cervical samples: a comparison of four genotyping methods. BMC Infect Dis 15:544. https://doi.org/10.1186/s12879-015-1281-5 CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Baldassarri R, Aronberg R, Levi AW, Yarbrough WG, Kowalski D, Chhieng D (2015) Detection and genotype of high-risk human papillomavirus in fine-needle aspirates of patients with metastatic squamous cell carcinoma is helpful in determining tumor origin. Am J Clin Pathol 143(5):694–700. https://doi.org/10.1309/ajcpcza4pszcfhq4 CrossRefPubMedGoogle Scholar
- 14.Pileggi C, Flotta D, Bianco A, Nobile CG, Pavia M (2014) Is HPV DNA testing specificity comparable to that of cytological testing in primary cervical cancer screening? Results of a meta-analysis of randomized controlled trials. Int J Cancer 135(1):166–177. https://doi.org/10.1002/ijc.28640 CrossRefPubMedGoogle Scholar
- 17.Manos MM, Ting Y, Wright DK, Lewis J, Broker TR, Wolinsky SM (1989) The use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells 7:209–214Google Scholar
- 18.Jacobs MV, de Roda Husman AM, van den Brule AJ, Snijders PJ, Meijer CJ, Walboomers JM (1995) Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes. J Clin Microbiol 33(4):901–905PubMedPubMedCentralGoogle Scholar
- 19.de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ (1995) The use of general primers GP5 and GP6 elongated at their 3′ ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 76(Pt 4):1057–1062. https://doi.org/10.1099/0022-1317-76-4-1057 CrossRefPubMedGoogle Scholar
- 20.Tornesello ML, Duraturo ML, Salatiello I, Buonaguro L, Losito S, Botti G, Stellato G, Greggi S, Piccoli R, Pilotti S, Stefanon B, de Palo G, Franceschi S, Buonaguro FM (2004) Analysis of human papillomavirus type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer. J Med Virol 74(1):117–126. https://doi.org/10.1002/jmv.20154 CrossRefPubMedGoogle Scholar
- 21.Biedermann K, Dandachi N, Trattner M, Vogl G, Doppelmayr H, More E, Staudach A, Dietze O, Hauser-Kronberger C (2004) Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue. J Clin Microbiol 42(8):3758–3765. https://doi.org/10.1128/jcm.42.8.3758-3765.2004 CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, Vollmer N, Menton S, Menton M, Dietz K, Wallwiener D, Kandolf R, Bultmann B (2004) Detection and typing of human papillomavirus by E6 nested multiplex PCR. J Clin Microbiol 42(7):3176–3184. https://doi.org/10.1128/jcm.42.7.3176-3184.2004 CrossRefPubMedPubMedCentralGoogle Scholar