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 Andrea1, Meini Genny2, Rossetti Barbara1, Bianco Claudia1,
Di Giambenedetto Simona3, Sighinolfi Laura4, Monno Laura5,
Castagna Antonella6, Capobianchi Maria Rosaria7,
d’Arminio Monforte Antonella8 and Zazzi Maurizio2
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
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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?
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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.
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Sampling strategy
Plasma
PBMC
52 (2-53)* wks
5
* Distances between time points represent medians (range)
PBMC
PBMC
57 (50-94) wks
120 (80-238) wks
3 wks
4,5
log copies/ml
4
3,5
3
HIV RNA
2,5
2
1,5
17 wks
1
0,5
0
HIV
diagnosis
cART start
time
fist VL
suppression
continued
suppression
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Results: patients characteristics and treatments
• 42 patients (33M/9F, median age 38, 48% heterosexual
contacts)
• Baseline CD4 351 cells/mm3 (IQR 188-520)
• Baseline CD8 1018 cells/mm3 (IQR 726-1297)
• Baseline VL 4.68 log10 cps/mL (IQR 4.30-5.01)
• CDC class C 7%
• cART type employed (first regimen):
– 2NRTI+1NNRTI: 28
– 2NRTI+PI: 11
– Other: 3
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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)
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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 137.3 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)
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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%
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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%
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
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. CozziLepri. 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 FP7 2007-2013 under the project "Collaborative HIV and Anti-HIV Drug
Resistance Network (CHAIN)" - grant n° 223131. Unrestricted Educational Grant: ViiV Healthcare to ADL and MZ
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona