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OP5-6 Evaluation of human papillomavirus (HPV) by quantification and typing in paired urine and cervical samples of women with abnormal cytology
S12
Oral presentations, 12th ESCV, Istanbul / Journal of Clinical Virology 46 Suppl. 1 (2009) S1–S14
OP5-5 Evaluation of a new strategy for cervix cancer screening in women who do not access to pap smear screening in West Brittany, using a urine test for human papillomavirus (HPV) detection on a large scale plateform combining EasyMag extractor and real-time PCR LightCycler system (the PAPU29 PHASE 1 study) C. Payan1 *, A. Tran1 , Y. Foll2 , C. Vallon2 , E. Poulhazan3 , K. Lacut3 , F. Charles4 , F. Herry5 , E. Postec6 , M. Collet6 . 1 D´epartement de Microbiologie-LUBEM EA3882, Centre Hospitalier Universitaire, Brest, France, 2 Association pour le d´epistage des cancers ADEC29, Brest, France, 3 Centre d’Investigation Clinique-INSERM0502, Centre Hospitalier Universitaire, Brest, France, 4 Laboratoire de Cytologie, Centre Hospitalier Universitaire, Brest, France, 5 Ecole Pratique des Hautes Etudes, St-Pol de L´eon, France, 6 Service de Gyn´ecologie-Obst´etrique, Centre Hospitalier Universitaire, Brest, France Background: In France, only 55% of women had a pap smear for cervix cancer screening owing to the lack of organized screening; this rate goes down to 35% in west Brittany. Aim: to evaluate a new strategy based on a rapid HPV-DNA assay in urine. Methods: HPV test was assessed in 1 mL of urine using the EasyMag extractor (BioMerieux) and real-time PCR LightCycler (Roche) systems allowing 48 automatic test samples per day. Urine samples were obtained between september 2008 and june 2009, from women of 25 to 65 years old who were non-responders to a previous invitation to pap smear around Morlaix, north city of Brittany. Positive samples were genotyped using the LiPA HPV test (Innogenetics). Results: EasyMag urine extraction showed a confident procedure (agreement 100%, cut-off at 15 copies, SD=1.4%) compared to viral mini kit (Qiagen) extraction previously described (Payan JCM2007). Among 5781 invited women, 312 (5.4%) accepted the pap smear test, 4036 denied (70%) and among them 1169 (30%) transmitted their urine sample to our lab; 19% were found HPV-positive and were invited to have a pap smear by their general practitioners. Conclusion: With about 5-fold increase screening compared to pap smear testing, our urine HPV test was evaluate using the EasyMag extractor and LightCycler platform, in a large cohort of women from general population with no Pap smear test over the last 3 years. Grants from the French Ligue contre le Cancer, technical help from Biom´erieux and Innogenetics. OP5-6 Evaluation of human papillomavirus (HPV) by quantification and typing in paired urine and cervical samples of women with abnormal cytology A. Ducancelle1 , S. Hantz2 , M.-C. Legrand3 , S. Alain2 , A. Beby-Defaux4 , C. Malbois5 , M. Avenel6 , M.-A. De Brux7 , F. Charles8 , F. Aspeele9 , L. Catala10 , P. Descamps10 , E. Postec11 , M. Collet11 , G. Agius4 , F. Lunel1 , C. Payan3 *. 1 Laboratoire de Virologie, CHU Angers, France, 2 Laboratoire de Virologie, CHU Dupuytren, Limoges, France, 3 D´epartement de Microbiologie-LUBEM, CHU Brest, France, 4 Laboratoire de Virologie, CHU La Milletrie, Poitiers, France, 5 Centre de Protection maternelle et infantile, Angers, France, 6 Centre antiv´en´erien, CHU Angers, France, 7 Laboratoire de Cytologie, CHU Angers, France, 8 Laboratoire de Cytologie, CHU Brest, France, 9 Centre d’Orthog´enie, CHU Angers, France, 10 Service de Gyn´ ecologie-Obstetrique, CHU Angers, France, 11 Service de Gyn´ecologie-Obstetrique, CHU Brest, France Aim: To evaluate a rapid consensus quantitative HPV DNA assay for the diagnosis of HPV infection in urine and cervical samples in a prospective multicentric (PAPU) study. Methods: Urine and cervical paired samples were collected from 250 women with abnormal cytology (mean age 36) between May 2004 and December 2007 at Angers, Brest and Limoges University Hospitals, and were assessed using a SybrGreen real-time PCR Mx4000 (Stratagene) and LightCycler (Roche) systems (Payan JCM2007). Positive samples were genotyped using the LiPA HPV test (Innogenetics). Results: HPV positive test was found in 143 samples (57.2%), 117 were positive in urine (sensitivity 82%, specificity 97%, agreement 87%). Mean viral loads in cervical and urine sample were respectively at 5.74 and 4.14 logDNAcopies/ml, with a range from 2.41 to 10.40 logs. High viral load was found in high grade lesions in cervical and urine samples (respectively 7.05 and 5.25 logs vs 4.80 and 3.99 logs in normal cytology, p<0.01). The same high risk HPV types were found in all paired positive samples, with a
majority of HPV16 (30%). Only 6 upon 30 urine samples were found positive with the Hybrid Capture(HC) II assay (Digene Corporation); negative results were obtained below 4 logs. Conclusion: Our HPV-DNA real-time PCR assay allows confident HPV detection and typing in urine that should facilitate the diagnosis of high risk HPV infection in patients with abnormal smears. HC-II test appears not adapted for HPV detection in urine samples. Grants from the French Ligue contre le Cancer OP5-7 Diversification of HIV-1 subtypes in Turkey – a country with a low HIV endemicity 1, ˙ K. Midilli1 *, G. Yilmaz2 , M.A. Ku¸skucu1 , S. Ergin1 , A. Istanbullu S. T¨urko˘glu1 , A. Kaygusuz1 , K. Alta¸s1 . 1 Microbiology and Clinical Microbiology Department, Cerrahpasa Faculty of Medicine, Istanbul, Turkey, 2 Microbiology and Clinical Microbiology Department, Yeditepe University Faculty of Medicine, Istanbul, Turkey In Turkey the first case of the HIV infection was described in 1985 and the number of the registered cases is stil below 3000. In a previous study based on partial env sequences almost 80% of HIV-1 subtypes in Turkey had been reported as subtype B. In this study, phylogenetic analysis of entire protease, partial polymerase and protease + partial polymerase sequuences of 98 patients referred between 2004 to 2009 in our laboratory for genotypic drug resistance analysis. The results are shown in the table. Genotype
B BF A1 AB AG AE F D C A K G
Protease 2004– 2007 2006
2008
2009
RT 2004– 2006
2007
2008
2009
GAG 2004– 2006
2007
2008
2009
15 − − − 2 2 2 − − − − −
15 − − − 3 1 2 1 1 5 1 −
12 − − − 2 1 − − 2 4 − 1
18 − − − − 1 1 − − 1 − −
25 − − − − 1 − − − − − −
17 − − − 1 6 3 − 2 − − −
13 − − − 1 5 − − 2 − − 1
10 6 2 2 − − 1 − − − − −
16 7 − 2 − 1 − − − − − −
10 5 6 2 1 − 3 − 2 − − −
9 3 − 1 1 − − − 2 5 − 1
23 − − − 2 1 − − − − − −
Despite to the low endemicity, ongoing diversification of the HIV 1 subtypes suggests that still these strains are mainly introduced via travel into our country or acquired abroad. OP5-8 Hexadecyloxypropyl-cidofovir (CMX001) BK virus (BKV) replication in primary renal tubular epithelial cells C.H. Rinaldo1 *, R. Gosert2 , H.H. Hirsch2 . 1 Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway, 2 Transplantation Virology, Institute for Medical Microbiology, University of Basel, Basel, Switzerland Background: There are no antiviral drugs with proven efficacy for polyomavirus-associated nephropathy or hemorrhagic cystitis in kidneyand bone marrow transplant patients, respectively. The nucleotide analogue cidofovir (CDV) is given intravenously but randomized clinical trials are either lacking or have failed to provide evidence of efficacy. Another caveat of CDV use is its pronounced nephrotoxicity which primarily affects renal tubular epithelial cells (RPTECs). The cidofovir derivate, hexadecyloxypropylcidofovir (CMX001), is taken orally and has a longer half-life than CDV. We have characterized the effects of CMX001 on BKV replication in RPTECs. Methods: RPTECs were infected with BKV(Dunlop). CMX001 was added before and 2 h postinfection (hpi). Cells and supernatants were harvested 24−72 hpi. BKV replication was examined by TaqMan assays, western blotting, IF staining and viability of RPTECs was examined by WST-1 assay, BrdU incorporation and a TaqMan assay. Results: CMX001 0.31mM reduced extracellular BKV loads by 90% 72 hpi. At this concentration we observed an approximately 20% reduction in BrdU incorporation and WST-1 activity. BKV entry and early expression was unaffected but BKV DNA replication reduced by 94% at 48 hpi. Late protein expression was 86% and 63% reduced 48 and 72 hpi. Conclusion: CMX001 inhibits BKV replication at the level of DNA replication. CMX001 0.31 uM gives a 90% reduction of extracellular BKV
Oral presentations, 12th ESCV, Istanbul / Journal of Clinical Virology 46 Suppl. 1 (2009) S1–S14
OP5-5 Evaluation of a new strategy for cervix cancer screening in women who do not access to pap smear screening in West Brittany, using a urine test for human papillomavirus (HPV) detection on a large scale plateform combining EasyMag extractor and real-time PCR LightCycler system (the PAPU29 PHASE 1 study) C. Payan1 *, A. Tran1 , Y. Foll2 , C. Vallon2 , E. Poulhazan3 , K. Lacut3 , F. Charles4 , F. Herry5 , E. Postec6 , M. Collet6 . 1 D´epartement de Microbiologie-LUBEM EA3882, Centre Hospitalier Universitaire, Brest, France, 2 Association pour le d´epistage des cancers ADEC29, Brest, France, 3 Centre d’Investigation Clinique-INSERM0502, Centre Hospitalier Universitaire, Brest, France, 4 Laboratoire de Cytologie, Centre Hospitalier Universitaire, Brest, France, 5 Ecole Pratique des Hautes Etudes, St-Pol de L´eon, France, 6 Service de Gyn´ecologie-Obst´etrique, Centre Hospitalier Universitaire, Brest, France Background: In France, only 55% of women had a pap smear for cervix cancer screening owing to the lack of organized screening; this rate goes down to 35% in west Brittany. Aim: to evaluate a new strategy based on a rapid HPV-DNA assay in urine. Methods: HPV test was assessed in 1 mL of urine using the EasyMag extractor (BioMerieux) and real-time PCR LightCycler (Roche) systems allowing 48 automatic test samples per day. Urine samples were obtained between september 2008 and june 2009, from women of 25 to 65 years old who were non-responders to a previous invitation to pap smear around Morlaix, north city of Brittany. Positive samples were genotyped using the LiPA HPV test (Innogenetics). Results: EasyMag urine extraction showed a confident procedure (agreement 100%, cut-off at 15 copies, SD=1.4%) compared to viral mini kit (Qiagen) extraction previously described (Payan JCM2007). Among 5781 invited women, 312 (5.4%) accepted the pap smear test, 4036 denied (70%) and among them 1169 (30%) transmitted their urine sample to our lab; 19% were found HPV-positive and were invited to have a pap smear by their general practitioners. Conclusion: With about 5-fold increase screening compared to pap smear testing, our urine HPV test was evaluate using the EasyMag extractor and LightCycler platform, in a large cohort of women from general population with no Pap smear test over the last 3 years. Grants from the French Ligue contre le Cancer, technical help from Biom´erieux and Innogenetics. OP5-6 Evaluation of human papillomavirus (HPV) by quantification and typing in paired urine and cervical samples of women with abnormal cytology A. Ducancelle1 , S. Hantz2 , M.-C. Legrand3 , S. Alain2 , A. Beby-Defaux4 , C. Malbois5 , M. Avenel6 , M.-A. De Brux7 , F. Charles8 , F. Aspeele9 , L. Catala10 , P. Descamps10 , E. Postec11 , M. Collet11 , G. Agius4 , F. Lunel1 , C. Payan3 *. 1 Laboratoire de Virologie, CHU Angers, France, 2 Laboratoire de Virologie, CHU Dupuytren, Limoges, France, 3 D´epartement de Microbiologie-LUBEM, CHU Brest, France, 4 Laboratoire de Virologie, CHU La Milletrie, Poitiers, France, 5 Centre de Protection maternelle et infantile, Angers, France, 6 Centre antiv´en´erien, CHU Angers, France, 7 Laboratoire de Cytologie, CHU Angers, France, 8 Laboratoire de Cytologie, CHU Brest, France, 9 Centre d’Orthog´enie, CHU Angers, France, 10 Service de Gyn´ ecologie-Obstetrique, CHU Angers, France, 11 Service de Gyn´ecologie-Obstetrique, CHU Brest, France Aim: To evaluate a rapid consensus quantitative HPV DNA assay for the diagnosis of HPV infection in urine and cervical samples in a prospective multicentric (PAPU) study. Methods: Urine and cervical paired samples were collected from 250 women with abnormal cytology (mean age 36) between May 2004 and December 2007 at Angers, Brest and Limoges University Hospitals, and were assessed using a SybrGreen real-time PCR Mx4000 (Stratagene) and LightCycler (Roche) systems (Payan JCM2007). Positive samples were genotyped using the LiPA HPV test (Innogenetics). Results: HPV positive test was found in 143 samples (57.2%), 117 were positive in urine (sensitivity 82%, specificity 97%, agreement 87%). Mean viral loads in cervical and urine sample were respectively at 5.74 and 4.14 logDNAcopies/ml, with a range from 2.41 to 10.40 logs. High viral load was found in high grade lesions in cervical and urine samples (respectively 7.05 and 5.25 logs vs 4.80 and 3.99 logs in normal cytology, p<0.01). The same high risk HPV types were found in all paired positive samples, with a
majority of HPV16 (30%). Only 6 upon 30 urine samples were found positive with the Hybrid Capture(HC) II assay (Digene Corporation); negative results were obtained below 4 logs. Conclusion: Our HPV-DNA real-time PCR assay allows confident HPV detection and typing in urine that should facilitate the diagnosis of high risk HPV infection in patients with abnormal smears. HC-II test appears not adapted for HPV detection in urine samples. Grants from the French Ligue contre le Cancer OP5-7 Diversification of HIV-1 subtypes in Turkey – a country with a low HIV endemicity 1, ˙ K. Midilli1 *, G. Yilmaz2 , M.A. Ku¸skucu1 , S. Ergin1 , A. Istanbullu S. T¨urko˘glu1 , A. Kaygusuz1 , K. Alta¸s1 . 1 Microbiology and Clinical Microbiology Department, Cerrahpasa Faculty of Medicine, Istanbul, Turkey, 2 Microbiology and Clinical Microbiology Department, Yeditepe University Faculty of Medicine, Istanbul, Turkey In Turkey the first case of the HIV infection was described in 1985 and the number of the registered cases is stil below 3000. In a previous study based on partial env sequences almost 80% of HIV-1 subtypes in Turkey had been reported as subtype B. In this study, phylogenetic analysis of entire protease, partial polymerase and protease + partial polymerase sequuences of 98 patients referred between 2004 to 2009 in our laboratory for genotypic drug resistance analysis. The results are shown in the table. Genotype
B BF A1 AB AG AE F D C A K G
Protease 2004– 2007 2006
2008
2009
RT 2004– 2006
2007
2008
2009
GAG 2004– 2006
2007
2008
2009
15 − − − 2 2 2 − − − − −
15 − − − 3 1 2 1 1 5 1 −
12 − − − 2 1 − − 2 4 − 1
18 − − − − 1 1 − − 1 − −
25 − − − − 1 − − − − − −
17 − − − 1 6 3 − 2 − − −
13 − − − 1 5 − − 2 − − 1
10 6 2 2 − − 1 − − − − −
16 7 − 2 − 1 − − − − − −
10 5 6 2 1 − 3 − 2 − − −
9 3 − 1 1 − − − 2 5 − 1
23 − − − 2 1 − − − − − −
Despite to the low endemicity, ongoing diversification of the HIV 1 subtypes suggests that still these strains are mainly introduced via travel into our country or acquired abroad. OP5-8 Hexadecyloxypropyl-cidofovir (CMX001) BK virus (BKV) replication in primary renal tubular epithelial cells C.H. Rinaldo1 *, R. Gosert2 , H.H. Hirsch2 . 1 Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway, 2 Transplantation Virology, Institute for Medical Microbiology, University of Basel, Basel, Switzerland Background: There are no antiviral drugs with proven efficacy for polyomavirus-associated nephropathy or hemorrhagic cystitis in kidneyand bone marrow transplant patients, respectively. The nucleotide analogue cidofovir (CDV) is given intravenously but randomized clinical trials are either lacking or have failed to provide evidence of efficacy. Another caveat of CDV use is its pronounced nephrotoxicity which primarily affects renal tubular epithelial cells (RPTECs). The cidofovir derivate, hexadecyloxypropylcidofovir (CMX001), is taken orally and has a longer half-life than CDV. We have characterized the effects of CMX001 on BKV replication in RPTECs. Methods: RPTECs were infected with BKV(Dunlop). CMX001 was added before and 2 h postinfection (hpi). Cells and supernatants were harvested 24−72 hpi. BKV replication was examined by TaqMan assays, western blotting, IF staining and viability of RPTECs was examined by WST-1 assay, BrdU incorporation and a TaqMan assay. Results: CMX001 0.31mM reduced extracellular BKV loads by 90% 72 hpi. At this concentration we observed an approximately 20% reduction in BrdU incorporation and WST-1 activity. BKV entry and early expression was unaffected but BKV DNA replication reduced by 94% at 48 hpi. Late protein expression was 86% and 63% reduced 48 and 72 hpi. Conclusion: CMX001 inhibits BKV replication at the level of DNA replication. CMX001 0.31 uM gives a 90% reduction of extracellular BKV