THE 6TH 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 F1-2, TRAN HONG T6., HO THI H4, PHAM VA4, NGUYEN L5, THONG LA3, TUAILLON E7, NGUYEN TA6, BUI DUC D3, 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)
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Outline
1. Background
2. Objectives
3. Methods
4. Results & Discussions
5. Conclusions
6. Recommendations
The 6th 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
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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
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Challenges for VL monitoring in decentralized area
• Several type of constraints:
– Technical
– Human
– Financial
Only reference laboratories able to provide VL measurements.
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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
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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
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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)
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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
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Study site and population:
• HIV-1 positive adults,
• Followed at Dong Da OPC in Hanoï
• Who agreed to participate
METHODS
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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
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Laboratory proceduresTraining VL DBS testing for laboratory staff
• 2 trainings for each technique
• performed by the manufacturer teams
• before the beginning of the study
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RESULTs Population description
• July 1st to October 9th, 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 category N (%)
Pre ART 19 (9.6)
1-6 months 34 (17.2)
> 6 months 51 (25.8)
Suspected treatment failure 94 (47.4)
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Roche technique
Plasma≥1000 cp/mL
<1000 cp/mL Total
DBS (FEV protocol)
≥1000 cp/mL 23 0 23
<1000 cp/mL 30 145 175
Total 53 145 198
Sensitivity 43.4% [29.8-57.7]
Specificity 100% [97.4-100]
Concordance 85.6%
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Roche technique
• Pearson correlation coefficient: r=0.81 (p<0.001)
Plasma Viral Load (log10 cp/mL)
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Abbott technique
Plasma≥1000 cp/mL
<1000 cp/mL Total
DBS
≥1000 cp/mL 42 8 148
<1000 cp/mL 3 145 50
Total 45 153 198
Sensitivity 93.3% [81.7-98.6]
Specificity 94.8% [90.0-97.7]
Concordance 94.4%
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Abbott technique
• Pearson correlation coefficient: r=0.91 (p<0.001)
Plasma Viral Load (log10 cp/mL)
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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
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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
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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]
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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
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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)
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CONCLUSIONS
• Study which aims at replicate routine condition of VL monitoring (delay between collection sample and VL measurement; condition of DBS storage, etc.)
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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
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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)
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Thank you for your attention