Presented at the 10 th Eu Meeting on HIV & Hepatitis, March 2012, Barcelona
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1 HIV-1 co-receptor tropism evolution in naïve patients undergoing successful ART: concordance of DNA vs RNA and triplicate versus singlicate population sequencing De Luca Andrea 1, Meini Genny 2, Rossetti Barbara 1, Bianco Claudia 1, Di Giambenedetto Simona 3, Sighinolfi Laura 4, Monno Laura 5, Castagna Antonella 6, Capobianchi Maria Rosaria 7, d Arminio Monforte Antonella 8 and Zazzi Maurizio 2 1 Academic Division of Infectious Diseases, University Hospital of Siena; 2 Department of Biotechnology, University of Siena; 3 Institute of Clinical Infectious Diseases, Catholic University, Rome; 4 Division of Infectious Diseases, Hospital of Ferrara; 5 Clinic of Infectious Diseases, University of Bari; 6 Clinic of Infectious Diseases, Hospital San Raffaele, Milan; 7 National Institute of Infectious Diseases L.Spallanzani, Rome;8 Clinic of Infectious Diseases, San Paolo Hospital, University of Milan
2 Background European Guidelines: gp120 V3 sequencing as a tool to assess HIV-1 co-receptor tropism: if plasma HIV RNA amplifiable: triplicate testing +G2P FPR 10% if plasma HIV RNA not amplifiable (low/undetectable VL): cellassociated HIV DNA triplicate testing +G2P FPR 10% Both on DNA and RNA single amplification: use G2P FPR 20% Tropism testing in VL suppressed useful for switch to CCR5 antagonists (toxicity/simplification): DNA tropism during HIV RNA suppression = baseline DNA/RNA? What is best testing strategy? How much is triplicate testing necessary?
3 Objectives To analyze the concordance between pre-cart HIV-1 RNA tropism and DNA co-receptor tropism after reaching VL suppression To determine, in a longitudinal study, the influence of time on VL suppression on tropism switch To establish the accuracy of singlicate versus triplicate testing on HIV-1 RNA and DNA tropism assignment using different interpretation thresholds
4 Patients and Methods ICONA patients naive, starting cart, achieving confirmed VL suppression (<50 cp/ml) Pre-cART plasma + PBMC available (same date) Additional PBMC samples at 2 time points during VL suppression (first and last available): VL<50 copies/ml at sampling and between the 2 PBMC samples Standard V3 population sequencing at all time points on DNA and at BL on RNA and DNA in triplicate: g2p FPR 10% Correlation analysis singicate/triplicate at different FPR RNA/DNA and BL/FU concordance analysis.
5 Sampling strategy Plasma PBMC PBMC PBMC 52 (2-53)* wks 57 (50-94) wks 120 (80-238) wks * Distances between time points represent medians (range) 5 4,5 3 wks 4 log copies/ml 3,5 3 2,5 2 1,5 17 wks HIV RNA 1 0,5 0 HIV diagnosis cart start time fist VL suppression continued suppression
6 Results: patients characteristics and treatments 42 patients (33M/9F, median age 38, 48% heterosexual contacts) Baseline CD4 351 cells/mm 3 (IQR ) Baseline CD cells/mm 3 (IQR ) Baseline VL 4.68 log 10 cps/ml (IQR ) CDC class C 7% cart type employed (first regimen): 2NRTI+1NNRTI: 28 2NRTI+PI: 11 Other: 3
7 Results: tropism genotyping Tropism test performed: 163 of 168 valid samples 482 of 489 (98.6%) amplified and sequenced Pre-cART tropism results: RNA non-r5:11/42; DNA non-r5:10/40; pre-cart RNA/DNA concordances: 40/40 (0 discordances)
8 Results: longitudinal tropism evolution Pre-cART RNA - first DNA after VL suppression: R5-nonR5: 2/31 (6.5%) during 43.7 PYFU (4.6 per 100 PYFU) nonr5-r5: 0/11 (0%) during 17.3 PYFU Pre-cART RNA - last DNA after VL suppression: R5-nonR5: 2/29 (6.9%) during PYFU (1.5 per 100 PYFU) nonr5-r5: 1/10 (10%) during 40.6 PYFU (2.5 per 100 PYFU) First - last DNA after VL suppression: R5-nonR5: 1/27 (3.7%) during 92.4 PYFU (1.1 per 100 PYFU) nonr5-r5: 2/12 (16.7%) during 30.4 PYFU (6.6 per 100 PYFU)
9 Accuracy of non-r5 detection by singlicate at different FPR on DNA or RNA: triplicate FPR10% as reference sensitivity specificity PPV NPV Singlicate FPR 10% ALL 88.8% 100% 100% 95.9% Singlicate FPR 20% ALL 95.5% 79.6% 64.1% 97.9% Singlicate FPR 10% RNA 81.9% 100% 100% 93.9% Singlicate FPR 20% RNA 100% 80.6% 64.7% 100% Singlicate FPR 10% DNA 91% 100% 100% 97% Singlicate FPR 20% DNA 94.1% 79.3% 64% 97.1%
10 Accuracy of non-r5 detection by singlicate at different FPR on DNA or RNA: triplicate FPR 5.75% as reference sensitivity specificity PPV NPV Singlicate FPR 10% ALL 94.4% 95.3% 85% 98.4% Singlicate FPR 20% ALL 94.4% 74% 50.7% 98% Singlicate FPR 10% RNA 100% 97% 88% 100% Singlicate FPR 20% RNA 100% 73% 47% 100% Singlicate FPR 10% DNA 92.8% 94.6% 83.8% 97.7% Singlicate FPR 20% DNA 92.8% 74.2% 52% 97.1%
11 Tropism discordances among triplicates of the same samples on the basis of FPR On all samples (n=162) With FPR 10%: 6/162 (3.7%) discordant tropism assignments 2 in plasma HIV RNA, 4 in PBMC HIV DNA With FPR 5.75%: 2/162 (1.2%) discordant tropism assignments 2 in PBMC HIV DNA
12 Conclusions Tropism on RNA/DNA pre-cart totally concordant Good concordance between pre-cart RNA tropism and DNA tropism at VL suppression Some shift towards non-r5 mostly occurring between pre-cart and VL suppression (residual replication?) suggests testing HIV DNA at suppression might detect more nonr5 than BL RNA Singlicate tropism genotyping with g2p FPR 10% shows sufficient concordance with triplicate On both RNA and DNA, particularly using FPR 5.75% as reference
13 Acknowledgements: ICONA Foundation Study GOVERNING BODY- M. Moroni (Chair). G. Angarano. A. Antinori. G. Carosi. R. Cauda. A. d Arminio Monforte. G. Di Perri. M. Galli. R. Iardino. G. Ippolito. A. Lazzarin. C.F. Perno. P.L. Viale. F Von Schlosser. SCIENTIFIC SECRETARY- A d Arminio Monforte STEERING COMMITTEE A. Ammassari. M Andreoni. A. Antinori. C. Balotta. P. Bonfanti. S Bonora. M Borderi. M.R. Capobianchi. A. Castagna. F. Ceccherini-Silberstein. A. Cozzi- Lepri. A. d Arminio Monforte. A. De Luca. M Gargiulo. C. Gervasoni. E. Girardi. M Lichtner. S. Lo Caputo. G Madeddu. F Maggiolo. S Marcotullio. L Monno. R. Murri. C. Mussini. M. Puoti. C. Torti STATISTICAL AND MONITORING TEAM A Cozzi-Lepri. I Fanti. T Formenti PARTICIPATING CLINICIANS AND CENTERS M. Montroni. A. Giacometti. A Costantini. A. Riva (Ancona); U. Tirelli. F. Martellotta (Aviano-PN); G. Angarano. L Monno. N. Ladisa. (Bari); F. Suter. F. Maggiolo (Bergamo); PL: Viale. G. Verucchi. B Piergentili. (Bologna); G. Carosi. G. Cristini. C. Torti. C. Minardi. D. Bertelli (Brescia); T. Quirino. C Abeli (Busto Arsizio); P.E. Manconi. P. Piano (Cagliari); J Vecchiet. K Falasca (Chieti); G Carnevale. S Lorenzotti (Cremona); L. Sighinolfi. D. Segala (Ferrara); F. Leoncini. F. Mazzotta. M. Pozzi. S. Lo Caputo (Firenze); G. Cassola. G Viscoli. A. Alessandrini. R. Piscopo. G Mazzarello (Genova); C. Mastroianni. V. Belvisi (Latina); P. Bonfanti. C Molteni (Lecco); A. Chiodera. P. Castelli (Macerata); M Galli. A. Lazzarin. G. Rizzardini. M. Puoti. A. d Arminio Monforte. AL Ridolfo. A Foschi. A Castagna. S Salpietro. S. Merli. L Carenzi. M.C. Moioli. P Cicconi. T Formenti (Milano); R. Esposito. C. Mussini (Modena); A Gori. V Pastore (Monza). N. Abrescia. A. Chirianni. M. De Marco. (Napoli); C. Ferrari. R Borghi (Parma); F Baldelli. B Belfiori (Perugia); G. Parruti. F Sozio (Pescara); G. Magnani. M.A. Ursitti (Reggio Emilia); M. Arlotti. P. Ortolani (Rimini); R. Cauda. M Andreoni. A. Antinori. G. Antonucci. P. Narciso. V Tozzi. V. Vullo. A. De Luca. M. Zaccarelli. L Gallo. R. Acinapura. P. De Longis. L Ceccarelli. R Libertone. M.P. Trotta. A Miccoli. (Roma); AM Cattelan (Rovigo); M.S. Mura. G Madeddu (Sassari); P. Caramello. G. Di Perri. G.C. Orofino. M Sciandra (Torino); E. Raise. F. Ebo (Venezia); G. Pellizzer. D. Buonfrate (Vicenza). The Icona Foundation Study is supported by unrestricted educational grants of Abbott. Bristol-Myers Squibb. Gilead Funding from the European Community's FP under the project "Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)" - grant n Unrestricted Educational Grant: ViiV Healthcare to ADL and MZ
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