1. THE 6 TH NATIONAL SCIENTIFIC CONFERENCE ON HIV/AIDS Evaluation of two techniques for viral load monitoring on DBS ANRS 12338 project Phase I - Laboratory evaluation phase (The MOVIDA Vietnam project: Improving access to viral load monitoring in HIV-infected patients on ART in decentralised area using Dried Blood spot ) TAIEB F 1-2, TRAN HONG T 6., HO THI H 4, PHAM VA 4, NGUYEN L 5, THONG LA 3, TUAILLON E 7, NGUYEN TA 6, BUI DUC D 3, DO THI N. 3, MADEC Y. 1 1.Emerging Diseases Epidemiology Unit-Institut Pasteur, Paris (France) 2.Direction de la Recherche Clinique et du Développement-Assistance Publique des Hôpitaux de Paris- Saint-Louis Hospital, Paris (France) 3.Vietnam Authority of HIV/AIDS Control (VAAC), Hanoi (Vietnam) 4.Hanoi School of Public Health- HIV/AIDS Prevention and Control department-Hanoi (Vietnam) 5.DONG DA OPC, Hanoi (Vietnam) 6.National Institute of Hygiene and Epidemiology- Virology laboratory-HIV/AIDS Department, Hanoi (Vietnam) 7.INSERM U1058 unit- Pathogenesis and Control of Chronic Infections-Virology department, Montpellier University Hospital, Montpellier (France)

2. The 6 th National Scientific Conference on HIV/AIDS Outline 1.Background 2.Objectives 3.Methods 4.Results & Discussions 5.Conclusions 6.Recommendations

3. The 6 th National Scientific Conference on HIV/AIDS Follow-up of patients on ART Essentially clinical and immunological Virological monitoring: –still scarce –especially in decentralized areas BACKGROUND

4. The 6 th National Scientific Conference on HIV/AIDS Benefit of VL monitoring: Early and specific detection of therapeutic failure Objective measure (and improvement) of adherence to ART Adapted change of ART Prevents HIV drug resistance acquisition

5. The 6 th National Scientific Conference on HIV/AIDS Challenges for VL monitoring in decentralized area Several type of constraints: –Technical –Human –Financial Only reference laboratories able to provide VL measurements.

6. The 6 th National Scientific Conference on HIV/AIDS Challenges for VL monitoring in decentralized area Plasma (gold standard) transfer is possible but: –Rapid RNA destruction at ambient temperature –Costly (cold-chain) –Infectious sample Patients followed in decentralized area (>50% of patients) have low or no access to VL monitoring

7. The 6 th National Scientific Conference on HIV/AIDS Advantage for VL monitoring Dried Blood Spots (DBS) At the HIV care site, DBS are: –Easy to perform –Easy to maintain and safe (sample not contagious) –Easy to transfer: No cold chain By the existing postal network

8. The 6 th National Scientific Conference on HIV/AIDS Advantage for VL monitoring Dried Blood Spots (DBS) At the virology laboratory (centralised): –No extra equipment needed –Capacity development of the personnel 2 techniques of VL measurement on DBS have been validated: –Nuclisens by Biomerieux® –Abbott® m2000rt Development of new techniques –Roche® FVE protocol –First results are very promising (Xiaoning Wu X et al. Poster at 2014 IAS conference)

9. The 6 th National Scientific Conference on HIV/AIDS Evaluation of VL measurement on DBS in routine practice To evaluate Sensitivity and Specificity of: – DBS vs. plasma (gold standard) – at the threshold of 1000 copies/mL. OBJECTIVES

10. The 6 th National Scientific Conference on HIV/AIDS Study site and population: HIV-1 positive adults, Followed at Dong Da OPC in Hanoï Who agreed to participate METHODS

11. The 6 th National Scientific Conference on HIV/AIDS Laboratory procedures Transfer of Whole blood (10 mL EDTA tube) from Dong Da OPC to NIHE DBS cards performed at NIHE laboratory (min of 15 days before VL measurement) Centrifugation and conservation (-80°C) of plasma on cryotubes (2 mL) Testing VL plasma and DBS samples by: –Roche: Cobas®AmpliPrep/Cobas® TaqMan HIV-1 Test v2.0 –Abbott: Abbott m2000rt

12. The 6 th National Scientific Conference on HIV/AIDS Laboratory procedures Training VL DBS testing for laboratory staff 2 trainings for each technique performed by the manufacturer teams before the beginning of the study

13. The 6 th National Scientific Conference on HIV/AIDS RESULTs Population description July 1 st to October 9 th, 2015: 198 patients enrolled 145 (73,3%) males Median [IQR] age: 38 years [34-41] Median [IQR] time on ART: 38 months [3-74] Patients categoryN (%) Pre ART19 (9.6) 1-6 months34 (17.2) > 6 months51 (25.8) Suspected treatment failure94 (47.4)

14. The 6 th National Scientific Conference on HIV/AIDS Roche technique Plasma ≥1000 cp/mL <1000 cp/mL Total DBS (FEV protocol) ≥1000 cp/mL 230 <1000 cp/mL 30145175 Total53145198 Sensitivity43.4%[29.8-57.7] Specificity100%[97.4-100] Concordance85.6%

15. The 6 th National Scientific Conference on HIV/AIDS Roche technique Pearson correlation coefficient: r=0.81 (p<0.001) Plasma Viral Load (log 10 cp/mL) DBS Viral Load (log 10 cp/mL

16. The 6 th National Scientific Conference on HIV/AIDS Abbott technique Plasma ≥1000 cp/mL <1000 cp/mL Total DBS ≥1000 cp/mL 428148 <1000 cp/mL 314550 Total45153198 Sensitivity93.3%[81.7-98.6] Specificity94.8%[90.0-97.7] Concordance94.4%

17. The 6 th National Scientific Conference on HIV/AIDS Abbott technique Pearson correlation coefficient: r=0.91 (p<0.001) Plasma Viral Load (log 10 cp/mL) DBS Viral Load (log 10 cp/mL

18. The 6 th National Scientific Conference on HIV/AIDS DISCUSSION Good correlation between plasma and DBS with both techniques Sensitivity is much better with Abbott technique than with Roche technique Specificity is slightly better with Roche technique than with Abbott technique

19. The 6 th National Scientific Conference on HIV/AIDS Sensitivity: Ability to detect virological failure False virological success ( i.e. VL <1000 cp/ml on DBS ) explanation: –Destruction of RNA Condition of conservation?:  room temperature same conditions for both techniques

20. The 6 th National Scientific Conference on HIV/AIDS Sensitivity: Ability to detect virological failure False virological success ( i.e. VL <1000 cp/ml on DBS ) explanation: –Destruction of RNA Delay of conservation? –Median [IQR] delay (days): »Roche: 23 [15-71] »Abbott: 27 [22-70]

21. The 6 th National Scientific Conference on HIV/AIDS Sensitivity: capacity to detect virological failure False virological success ( i.e. VL <1000 cp/ml on DBS ) explanation: –Quantity of whole blood used? Roche: 1 spot of 70µL Abbott: 2 spots of 50µL

22. The 6 th National Scientific Conference on HIV/AIDS Specificity: Ability to detect virological success False virological failure ( i.e. VL >1000 cp/ml on DBS ) explanation: –Dried Blood Spot (whole blood) –Over estimation of VL: Amplification of proviral DNA (Monleau M, et al. J. Clin. Microbiol. 2009 and J. Antimicrob. Chemother. 2010)

23. The 6 th National Scientific Conference on HIV/AIDS CONCLUSIONS Study which aims at replicate routine condition of VL monitoring (delay between collection sample and VL measurement; condition of DBS storage, etc.)

24. The 6 th National Scientific Conference on HIV/AIDS Dried Blood Spot can overcome challenges to VL monitoring Is available immediately Allow to perform a large panel of analyze (Drug resistance genotyping, HCV-HBV serology; HCV VL (current studies). Better Sensitivity with Abbott technique than with Roche technique Sensitivity is the most important criterion in our opinion Indication to switch the treatment: 2 VL > 1000 cp/mL

25. The 6 th National Scientific Conference on HIV/AIDS RECOMMENDATIONS Further studies in “real life” to validate similar results Studies of other elution protocol with DBS Use of DBS for VL monitoring in decentralized area (eg: mountain area)

26. The 6 th National Scientific Conference on HIV/AIDS Thank you for your attention