A.M.J. Wensing, MD, PhD

1. Implementation of resistance testing on dried blood spots enables individual patient management in rural South-African Setting A.M.J. Wensing, MD, PhD

2. Introduction • Antiretroviral programmes have delivered HAART to 6,6 million patients in resource-limited settings • Monitoring is conducted by clinical symptoms, CD4-counts and, if available, viral load • Overall, effective viral suppression is achieved1 resulting in health improvement and extension of life 1. Barth et al. Lancet InfectiousDiseases 2010

3. Ndlovu Medical Center • ARV programme initiated in 2003: First-line ART: NNRTI, 3TC with (d4T)orAZT • Virologicalmonitoring every 6 months, yearly once viral suppression has been achieved • Viral suppression rate (<50 cp/ml) is ca 80% • After initial virological suppression ca 20% of patients experience a viral rebound (> 1000 cp/mL) • Upon repeated testing: resupression or switch • Ongoingviraemia during first-line ART has been documented in a limited group of patients

4. Accumulation of drug resistance observed during retrospective testing baseline T1 + 36% T2 + 15% + 76% 47 54 72 98 25 44 Barth et al. Antiviral Therapy in press

5. Obstacles to resistance testing in RLS • In case of therapy failure resistance testing is not generally available in remote settings due to: • High overall costs • Expensive equipment and limited after sales support • Use of plasma, which requires cold-chain maintenance • Insufficient space for appropriate laboratory flow to prevent contamination • Unreliable power supply can interrupt procedures, damage equipment and compromise sample integrity • Unreliable reagent supply • Shortage of skilled laboratory workers • Limited access to expertise for interpretation of results

6. Pilot Project • Pilot-project implementing resistance testing on dried blood spots (DBS) as routine procedure • Eligibility: patients experiencing viral rebound after initial viral suppression • DBS are prepared locally from whole blood. • Once dried DBS are no longer infectious they can be shipped at room temperature by airmail to a reference laboratory.

7. Test and report procedure • Inexpensive in house assay was modified for use of DBS • Nucleic acid is eluted from two 50ul spots using Nuclisenslysisbuffer • Nucleic acid is extracted using Minimag, amplified by nested RT-PCR for PR-RT and sequenced • Individual patient reports • Include susceptibility ranking based on freely available algorithms • Expert advice is added by a clinical virologist based on observed resistance patterns, treatment history and local drug availability • Final report is provided by email within 3 weeks

8. Email Report

9. Patient Characteristics 1st line 2nd line Total Number of patients 56 28 84 Age, median 36 34 36 Female gender 67% 71% 68% Pre-therapy CD4, median 74 89 76 CD4 at DBS time 215 224 218 Log HIV-RNA at DBS time 4,2 4,2 4,2 Time on HAART, yr 4,2 3,4 4,0 Time on PI, yr - 1,7 DBS succes rate 96% 93% 95% DBS succes rate 100% 100% 100% when VL >400

10. Resistance Patterns observed in DBS %

11. Extent of resistance was not related to therapy duration = 5 18 12 11 8 9 Mediannumber of mutations per year of treatment

12. In Summary • Resistance testing on DBS was successfully implemented as a routine clinical procedure in a rural setting • Resistance testing using in house methods enables optimized genotyping of DBS and reduces processing costs • Use of DBS sampling enables access to resistance testing even in rural settings

13. Acknowledgements: Collaborations • NdlovuMedical Center • Hugo Tempelman • Peter Schrooders • Mariette Slabbert ARTA University Medical Center Utrecht • Andy Hoepelman www.umcutrecht.nl/virusdiagnostics

14. UMC Utrecht, Antiviral Escape Group