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Field Study of the Utility of Dried Blood Spots (DBS) for HIV-1 Drug Resistance (HIVDR) Genotyping

Field Study of the Utility of Dried Blood Spots (DBS) for HIV-1 Drug Resistance (HIVDR) Genotyping. Storage for 2 Weeks and Shipping at Ambient Temperature Has No Effect on Genotyping Efficiency. Chris Parry MRC/UVRI Research Unit on AIDS. ART Roll Out.

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Field Study of the Utility of Dried Blood Spots (DBS) for HIV-1 Drug Resistance (HIVDR) Genotyping

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  1. Field Study of the Utility of Dried Blood Spots (DBS) for HIV-1 Drug Resistance (HIVDR) Genotyping Storage for 2 Weeks and Shipping at Ambient Temperature Has No Effect on Genotyping Efficiency Chris Parry MRC/UVRI Research Unit on AIDS

  2. ART Roll Out • Antiretroviral therapy (ART) continues to be rolled out in developing countries • Increased ART coverage means increased risk of drug resistance • HIV Drug Resistance monitoring in parallel to ART roll out • Testing limited (~27 WHO accredited labs)

  3. WHO HIVDR Laboratories This map is an approximation of actual country borders December 2011

  4. Samples for HIV drug resistance testing • Gold standard is plasma stored at -80oC • Need lab and staff to process whole blood • Freezer storage (ideally -80oC) • Transport frozen (ideally on dry ice) • Need for more user-friendly & cost effective alternative Dried Blood Spot (DBS)

  5. Optimal DBS storage and shipping conditions for HIV DR? • Samples (105) from patients failing ART in Uganda • Plasma (stored -80oC and shipped dry ice) • DBS cards (x4) from venous blood • 5th DBS card from finger prick • DBS • Stored ambient temp 2 or 4 weeks before transfer to -80oC • Shipped ambient temp or on dry ice to CDC lab (Atlanta) • Shipping temp monitored • Compared resistance testing results from DBS stored and shipped at different temperatures to plasma

  6. Sample Details *ARV drug use: AZT (98%), 3TC or FTC (100%), TDF (28%), ABC (4%), NVP (60%), EFV (20%), LPV/r (13%), IDV/r (1%)

  7. Genotyping Result * ** * p=0.03 vs group 3 ** p= 0.002 vs group 3 (finger prick)

  8. Shipping details

  9. Effect of Temp During Shipment * p=0.003 vs Shipment 2 Only 4 shipments with temp data and 10 or more samples included in analysis

  10. Nucleotide Sequence & DRM Identity vs Plasma *T-test P<0.05 vs. group 1 Mean % Nucleotide identity vs. Plasma was not significantly lower than 99.0% for any group Mean % DRM similarity vs. Plasma was not significantly lower than 99.5% for any group

  11. Amplification Summary • Compared to plasma no significant reduction in amplification efficiency for DBS stored at AT for 2 weeks and shipped at AT • A small but statistically significant reduction was observed in DBS stored at AT for 4 weeks and shipped at AT • Genotyping rate from finger prick DBS stored at AT for 2 weeks and shipped at AT was reduced compared to plasma or DBS made from EDTA blood stored at AT for 2 weeks and shipped at AT • Genotyping rate was highest in the shipment with the highest viral load, and was lower in shipments with maximum temperature >30°C

  12. Sequence Summary • Subtypes A (60), C (3), and D (40) were represented • Nucleotide sequence identity from DBS was high, except for finger prick • Resistance-associated mutation similarity to plasma was high • No statistically significant differences in resistance-associated mutations between DBS groups

  13. Conclusion • DBS stored dry at AT for 2 weeks • Ambient temp shipment of previously frozen DBS -are suitable for HIV genotyping • DBS from finger prick lower success rate • DBS genotyping rate positively associated with VL and negatively associated with duration of AT shipment and temperature.

  14. Acknowledgments S Mwebaza (Mildmay Uganda) R Batamwita (Mildmay Uganda) F Lyagoba (MRC/UVRI) B Magambo (MRC/UVRI) P Kaleebu (MRC/UVRI) N Parkin (Data First Consulting) M Jordan (WHO) S Bertagnolio (WHO) N Bbosa (CDC Uganda) R Downing (CDC Uganda) K Diallo (CDC Atlanta) J DeVos (CDC Atlanta) C Yang (CDC Atlanta) All patients and clinical staff who helped in collecting patient samples

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