BackgroundThe aims of this study were to detect HPV E6/E7 mRNA expression in women with high-risk genotypes (HPV-16, -18, -31, -33 and -45) analysing its relationship with tissue pathology and 2) 2-year follow-up of E6/E7 mRNA tested group.MethodsOur samples were genotyped and classified by pathologists according to Bethesda system. After RNA extraction, E6/E7 oncogene mRNA detection was performed by NucliSens® EasyQ® HPV v1 Test (bioMérieux).ResultsThe results of the present study showed that E6/E7 mRNA positivity rate was 68.29 % in women tested once and 69.56 % in women tested twice. According to tissue pathology, all samples with high-grade lesions were positive for mRNA. Among women with low-grade lesions varied over the years from 89.28 to 84 % in women tested once and from 77.77 to 70 % in tested twice. Among women without lesion, positivity rate maintained in women tested once (from 50 to 41.38 %) and decreased in tested twice, from 63.63 to 44.44 %. Regarding lesion evolution, mRNA positivity was higher in women with lesion progression (53.13 %) and in women with positive results in two tested samples (83.33 %).ConclusionHPV E6/E7 mRNA detection may be an effective screening test and biomarker for cervical cancer in women infected with these five genotypes. Nonetheless, further studies are needed to standardize as routine triage test.
BackgroundHuman papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, however to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease.Nowadays, mRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. We aimed to validate a method for RNA extraction, detect HPV mRNA expression and, assess the relationship between E6/E7 mRNA expression and pathology of patients’ lesions and progression.MethodsThis study included 50 specimens that had been previously genotyped as HPV16, 18, 31, 33 and/or 45. Cervical swabs were extracted with three different RNA extraction methods -Nuclisens manual extraction kit (bioMérieux), High Pure Viral RNA Kit (Roche) and RNeasy Plus Mini kit (Qiagen)-, and mRNA was detected with NucliSens EasyQ HPV version 1 test (bioMérieux) afterwards. Association of oncogene expression with pathology and lesion progression was analyzed for each extraction method.ResultsE6/E7 mRNA positivity rate was higher in samples analyzed with bioMérieux (62%), followed by Roche (24%) and Qiagen (6%). Women with lesions and lesion progression showed a higher prevalence of viral RNA expression than women that had not lesions or with lesion persistence. While bioMérieux revealed a higher sensitivity (77.27%), Roche presented a higher PPV (75%) and an increased specificity (89.28%).ConclusionsExtraction methods based on magnetic beads provided better RNA yield than those based in columns. Both Nuclisens manual extraction kit (bioMérieux) and High Pure Viral RNA Kit (Roche) seemed to be adequate for E6/E7 mRNA detection. However, none of them revealed both high sensitivity and specificity values. Further studies are needed to obtain and validate a standard gold method for RNA expression detection, to be included as part of the routine cervical screening program.
Aims: The aims of the study were (1) to characterize the genetic variability of human papillomavirus (HPV) genotype 16 in the E6 region when this genotype is present in multiple infection samples, (2) to assess the prevalence of variants in our region and (3) to analyze the relationship between variants, patients' ages and pathology. Methods: The Clinical Microbiology and Infection Control Department analyzed samples which were positive for genotype 16 and other genotypes from 2007 to 2013. Variants were assigned to European, Euro-German, Asian, Asian-American or African lineage by sequence analysis. The relationship among variants, age and different types of lesion was studied. Results: In HPV-16 sequence analysis, the European variant was detected in 85.10% of samples, the Asian-American in 7.80%, the African in 4.25% and the Euro-German in 2.83% of specimens. Sequence genetic variability showed 16 nucleotide substitutions. Moreover, non-European variants were mainly found in old women and in isolates from high-grade squamous intraepithelial lesions since European variants were mainly detected in negative cytologies. Conclusion: Multiple infections may take effect on nucleotide substitution and the appearance of recombinant samples. Single gene analysis makes it impossible to detect recombination which has a great influence on drug response and vaccine strategies. Thus, E6 gene analysis would be enough to identify HPV-16 intratypic variants but not to confirm the results.
Background/Objectives Human papillomavirus type 16 (HPV 16) is the primary aetiology of cervical cancer.Risk factors associated to develop of malignant lesions include: infection persistence, specific HPV 16 variants and multiple infections presence.We had characterised the genomic variability of E6, E7 and L1 genes in HPV 16 multiple infection patients samples and analysed the relationship between intratypic variants and lesion grade. Methods HPV 16 multiple infection samples were amplified with three region type-specific primers and amplicons were sequenced using the "Big Dye Terminator Cycle Sequencing kit".Sequences were aligned using Edit Sequence Alignment Editor and ClustalW, and compared with Genbank reported reference sequences: European (E), African (AF1 and AF2) and Asian-American (AA).Lesions were divided as negative, low-grade (L-SIL) or high-grade (H-SIL). Results HPV 16 multiple infections were identified in 125 samples and 78 of them were analysed for intratypic variations: 72 E variants (92.3%), 4 AA variants (5.1%), one AF1 (1.3%) and one AF2 variant (1.3%).In E6 region, missense mutations (A104del and T350G) were defined in 59% and 41% of samples. In E7 region, a mainly synonymous variation (G849A, 41.33%) was detected. In L1 region, nonsynonymous replacements were only identified: 6901insCAT (30%), 6902 insATC (65.7%) and GAT6951del (97.1%).European variants were mainly detected in samples with no lesion while non-european variants were only found in H-SIL or L-SIL. Conclusions E6, E7 and L1 genes are useful to determinate among E, AA and AF1/AF2 variants. Non-european variants are also present in our population.Nucleotide variations different to define variants must be studied owing to their potential impact on pathogenesis. T350G nucleotide substitution is associated with elevated risk of cervical carcinomas. These variations should be taken into consideration.Funding: S-PC11BF002 project (Saiotek, Department of Industry, Basque Government). molecular tyPing of Treponema pallidum from an ongoing syPHilis outBreak in Denmark
Giza papilomabirusa (HPV) 120 genotipo baino gehiagoz osaturiko birus taldea da. Genotipo horien guztien artean 40 bat inguruk gaitasun onkogenikoa dute eta minbizia sor dezakete gorputzeko hainbat ataletan:orofaringea, bulba, zakila edo umetoki-lepoa. Azken minbizi mota honek urtero 250.000 emakumeren heriotza eragiten du. Halaber, kontuan izan behar da HPV genotipo guztiek ez dutela onkogenikotasun maila bera aurkezten. 16 eta 18 genotipoak, adibidez, umetoki lepoko minbizi kasuen % 75etan aurkitu dira. Ikerketa lan honetan minbizia garatzeko arrisku altua duten giza papilomabirus (HPV) genotipoen populazio-baheketa egin dugu gure inguruko emakume talde batean (Bilbo, Euskal Herria), birus honen nagusitasuna eta genotipo desberdinen arteko banaketa ezagutzeko. Gure ikerketa honetan, ikusi da birus honen arrisku altuko genotipoen intzidentzia gure inguruan % 10,63 dela eta HPV 16 eta 18ak sortutako infekzioak umetoki-lepoko epitelioan lesioak sortzeko gaitasun altuagoa dutela; hain zuzen, HPV 16 duten emakumeen % 56,25ak lesioren bat garatu dute. Gainera, egiaztatu ahal izan da emakume gazteei nagusiki eragiten dien birusa dela; 30 urtetik beherako emakumeetan birusaren nagusitasuna % 24,87koa izan da, 30 eta 50 urte arteko emakumeetan % 8,37koa eta 50 urte baino nagusiagoak diren emakumeetan % 3,36koa. Azkenik, behatu izan da infekzio anitzek gaitasun handia dutela umetoki lepoko epitelioan lesioak sortzeko, batez ere 16 genotipoa arrisku altuko beste genotipo batzuekin batera daudenean. Gainera, infekzio anitzak emakume gazteetan baizik ez dira agertzen (batez besteko adina 28,89 urte).
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