Viral load of episomal and integrated forms of human papillomavirus type 33 in high‐grade squamous intraepithelial lesions of the uterine cervix

Abstract

The association between total and integrated HPV-33 DNA loads and high-grade squamous intraepithelial lesions (HSIL) of the uterine cervix was investigated. Of 5,347 women recruited in 4 studies, 89 (64 without SIL, 7 low-grade SIL (LSIL), 15 HSIL, 3 unknown grade) were infected by HPV-33. HPV-33 E6, HPV-33 E2 and b-globin DNA were measured with real-time PCR that allowed to assess total (E6), episomal (E2) and integrated (E6-E2) HPV-33 viral loads. HPV-33 E6/E2 ratios 2.0 suggesting the presence of integrated HPV-33 were obtained for 28.6% (n 5 18) of women without SIL and 21.4% (n 5 3) of women with HSIL (p 5 0.74). Although median viral loads were similar, there was a trend toward having a greater proportion of women with HSIL in the fourth quartile (

Influence of human papillomavirus type 16 (HPV-16) E2 polymorphism on quantification of HPV-16 episomal and integrated DNA in cervicovaginal lavages from women with cervical intraepithelial neoplasia

“…One of the latter methods, the detection of integrated papillomavirus sequences-PCR, found integrated HPV-16 in all 11 samples that contained mixed or integrated HPV-16 forms by real-time PCR and tested negative for all 20 samples that did not contain integrated HPV-16 (De Marco et al, 2007). We also confirmed by real-time PCR the presence of integrated HPV-33 forms with restricted-site PCR (Khouadri et al, 2007). Complete concordance between real-time PCR and a combination of Southern blot and 2D-gel electrophoresis was obtained on eight specimens only containing episomal forms and four specimens with mixed HPV-16 forms (Nagao et al, 2002).…”

“…The integration status of HPV-16 was assessed by comparing the levels of HPV-16 E2 and E6 genes and was expressed as the E6/E2 ratio. The integrated HPV-16 viral load was calculated by subtracting the copy numbers of E2 (episomal) from the copy numbers of E6 (integrated and episomal) for specimens with an E6/E2 ratio .2.0, as discussed previously (Fontaine et al, 2005a;Khouadri et al, 2007).…”

Low-risk human papillomavirus type 6 DNA load and integration in cervical samples from women with squamous intraepithelial lesions

“…8 Amplification of the internal control did not show any inhibition in these samples as measured by a signal corresponding to at least 80% of the expected signal. 8 HPV-6 DNA and ␤-globin DNA were quantitated with real-time PCR assays targeting E6 and E2, as described previously.…”

“…8 HPV-6 DNA and ␤-globin DNA were quantitated with real-time PCR assays targeting E6 and E2, as described previously. 8 Two microliters of each processed sample was tested in duplicate in each HPV-6 E6 and E2 assays, and for ␤-globin DNA. 8 Cycle thresholds were compared to those of serial ten-fold dilutions of an HPV-6 plasmid in DNA extracted from 10 4 human fibroblasts.…”

Human Papillomavirus Infection and Transmission Among Couples Through Heterosexual Activity (HITCH) Cohort Study: Protocol Describing Design, Methods, and Research Goals (Preprint)

“…The PCR sequencing method used primers flanking a segment of the long control region of HPV [18][19][20][21]. All genital specimens found to be HPV-DNA positive for types 16, 18, 31, 33, 45 and 52 were retested by a quantitative, real-time PCR to measure type-specific viral load using specific primers [22][23][24].…”