Dried Blood Spots (DBS) for HIV-1 viral load and Drug Resistance
Transcription
Dried Blood Spots (DBS) for HIV-1 viral load and Drug Resistance
5èmes journées du Site ANRS-Cameroun Yaoundé, 3-4 juin 2013 ANRS-12235 Le sang total séché sur papier filtre, DBS (Dried Blood Spot), est-il utilisable pour le suivi virologique en routine des patients VIH+ traités et suivis dans les programmes nationaux : étude multicentrique en Afrique et en Asie du Sud-Est AC11/AC12 ANRS: Groupe Résistance au Sud Coordination: M. Peeters/ML. Chaix ANRS-12235: Coordination: A. Ayouba/A. Aghokeng MEB: M. Monleau Biostatistiques: S. Eymard-Duvernay Context and Objectives Context Increasing number of patients on HAART in developing countries; limited access to biological monitoring for those located far from specialized laboratories; Main objective To evaluate the reliability of DBS to detect virological failure and drug resistance mutations in HIV infected patients in real-life conditions and following routine processes in resource-limited settings which are diverse in terms of climate characteristics with different ambient temperature and humidity level, but are also different in terms of available infrastructures and program functioning. Specific Objectives 1. Can DBS replace plasma for HIV viral load testing, regardless the viral load system used and the setting? 2. Are DRM profiles observed from plasma and DBS similar? 3. How many failing patients with DRM will be missed if DBS were used for HIV-1 genotyping? Methods (1): Study Sites • Burkina-Faso (Dr D. KANIA) – • Cameroun (Dr A. AGHOKENG) – • 3 sites de recrutement: Biyemassi, Cité Verte et Nkolndongo Sénégal (Pr C. TOURE-KANE) – • 1 site de recrutement: hôpital de jour de Sano-Soro de Bobo-Dioulasso 5 sites de recrutement (CTA, CRCF, CPS, HALD, Roi Baudoin) Thaïlande (Dr N.NGO-GIANG HUONG) – 5 sites de recrutement (Mae Chan Hospital, Chonburi Hospital, Hat Yai hospital, Maharaj Nakornratchasrima Hospital, Ratchaburi Hospital) • Togo (Dr A. Yaoste DAGNRA) – 5 sites de recrutement (CHU TOKOIN, Association EVT, Association CRIPS, Association AMC, Asprofem) • Vietnam (Dr L. Truong XUAN) – 3 sites de recrutement (OPC 1, OPC2, OPC4) Methods (2): STUDY FLOWCHART Blood samples from 1,621 adult patients receiving first line ART for 24 months were used to prepare in parallel plasma aliquots and DBS DBS Plasma -2-4 weeks at room temperature -Transfer to -20°C until use Nucleic acids extraction methods: - Qiagen viral RNA in Labs A and F - m2000rt, Abbott in Labs B, C, D - MiniMag, Biomérieux in Lab E Nucleic acids extraction methods: - m2000rt, Abbott in Labs B, C,D - MiniMag, Biomérieux in Labs A, E, F HIV-1 VL quantification by: -Generic Viral load (Biocentric) in Labs A & F -m2000rt (Abbott) in Labs B, C, D -EasyQ (Biomerieux) in Lab E If VL ≥1,000 copies/mL in Plasma and ≥5,000 in DBS amplification and sequencing of reverse transcriptase (RT) by nested RT-PCR. (ANRS protocol). Results(1): FEASIBILITY Parameter Mean [Interquartile Range] Ambient T° during DBS preparation (°C) 24 [22-26] 10 [4-18] 7 [0-9] 24 [-40-28] 1 [0-1] DBS drying (hours) DBS stockage on site (days) T° During transfer to Ref lab. (°C) Transfer duration (days) Results (2): Sensitivity and specificity to detect virological failure (>1,000 copies/ml) in DBS compared to plasma Viral load Assay N Abbott 173 Lab B Lab C Lab D Generic VL (Biocentric) Lab A Lab F 60 Plasma VL >1,000 cp/ml False Negative* Sensitivity [IC95%] False Positive** Specificity [IC95%] PL-DBS Pairs with detectable VL Nb diff < 0.5 log (%) 24 0 100 1 97.2 26 24 (40%) 53 13 [85.8-100] 0 (25%) 60 12 (20%) 100 7 [75.3-100] 3 75.0 (92%) [85.5-99.9] 82.5 18 [67.2-92.7] 1 [42.8-94.5] 97.9 12 (67%) 10 8 (80%) [88.9-99.9] 118 60 16 1 (27%) 58 23 93.8 17 [69.8-99.8] 2 (40%) 91.3 61.4 23 (70%) [45.5-75.6] 14 [72.0-98.9] 60.0 16 37 18 (49%) [42.1-76.1] EasyQ (Biomerieux Lab F 91 67 (74%) 10 85.1 [74.3-92.6] 1 95.8 [78.9-99.9] 76 28 (37%) *False Negative : VL >1,000 cp/ml in Plasma but <1,000 cp/ml in DBS. **False Positive : VL <1,000 cp/ml in Plasma but >1,000 cp/ml in DBS DBS VL (Log10 copies/ml) Results (3): Correlation between PL and DBS VL. Lab A Generic VL 8 7 6 5 4 3 2 8 7 6 5 4 3 2 (r=0.91) Lab B m2000rt 8 7 6 5 4 3 2 (r=0.98) Lab C m2000rt 8 7 6 5 4 3 2 (r=0.99) Depends on: 2 3 4 5 6 7 8 2 3 4 5 6 7 8 2 3 4 5 6 7 8 Lab E EasyQ Lab D m2000rt Lab F Generic VL (r=0.82) 2 3 4 5 6 7 8 8 7 6 5 4 3 2 8 (r=0.95) 7 (r=0.84) 6 5 4 3 2 2 3 4 5 6 7 8 2 3 4 5 6 7 8 Plasma VL (Log10 copies/ml) VL assay used Lab experience Results (5): Can DBS replace plasma for genotypic drug resistance testing: Are nucleotides sequences observed from plasma and DBS identical? 45 number of samples 40 Plasma vs DBS nucleotide sequences 35 30 <1% nt difference 1-1.99% nt difference 2-2.99% nt difference 3-3.99% difference >4% nt difference 25 20 15 10 5 0 lab A N=11 Lab B N=20 Lab C N=11 Lab D N=3 Lab E N=38 Lab F N=15 Total N=98 >80% of PL/DBS sequences pairs presented less than 3% nucleotides differences Results (6): Can DBS replace plasma for genotypic drug resistance testing: Are DRM profiles observed from plasma and DBS similar? Genotyped Any DRM Identical DRM profile Discordant DRM profile DRM in Plasma NOT DBS DRM in DBS NOT Plasma Different DRM profiles Plasma 98 90 DBS 98 81 75 23 10 3 10 Results (7): Quality control of NT sequences of discordant samples: Origin of discordant DRM profiles E-2558DBS E-2558PL F-701DBS F-701PL D-2149DBS D-2149PL C-1179DBS C-1179PL F-493DBS F-493PL F-307DBS F-307PL F-451DBS F-451PL F-622DBS F-622PL F-600DBS F-688DBS F-600PL F-704DBS F-252DBS F-252PL F-704PL F-688PL B-3175DBS B-3175PL C-2423DBS C-2423PL A-1224DBS A-1224PL 0.01 E-2292DBS E-2292PL B-1320DBS B-1320PL B-3161DBS B-3161PL C-4471DBS C-4471PL E-3120DBS E-3120PL B-2129DBS B-2129PL E-2055DBS E-2055PL E-3341DBS E-3341PL Some sequences obtained from Plasma and DBS of the same samples are different Results (8): How many drug resistant samples were not detected on DBS? Plasma study VL DRM in FN* site >1,000 DBS VL <1,000 DBS samples Lab Lab Lab Lab Lab Lab A B C D E F 16/60 24/60 13/53 12/60 67/91 23/58 1/16 0/24 0/13 3/12 10/67 2/23 0/1 2/3 10/10 2/2 Discordant DRM Plasma/DBS 1/11 4/20 3/11 (1 no DRM at all) 1/3 5/38 (1 no DRM at all) 9/15 (8 no DRM at all) 14/16 samples with DBS VL<1,000 cp/ml harbored HIV-1 with DRM 8/23 samples with discordant results showed no DRM. Conclusion • The present study shows that DBS can be an alternative to plasma for the follow-up of HIV-1 infected patients in routine settings; • Performances depend on: – VL assay characteristics ; • Detection of proviral DNA or not; – Redefine VL threshold for some assays? • Viral diversity; – Staff training • Necessity of staff training for HIV-1 genotyping using DBS and of external Quality Control; Acknowledgements The members of the AC11/AC12 “Résistance dans les pays du Sud” study group The patients who participated to this study - B. Bazin - C. Rekacewicz - G. Collin - M. Peeters - E. Delaporte