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 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)

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

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

Optimal DBS storage and shipping conditions for HIV DR? Samples (105) from patients failing ART in Uganda – Plasma (stored -80 o C and shipped dry ice) – DBS cards (x4) from venous blood – 5 th DBS card from finger prick DBS – Stored ambient temp 2 or 4 weeks before transfer to -80 o C – 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

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%)

Genotyping Result * ** * p=0.03 vs group 3 ** p= vs group 3 (finger prick) -80 o C AT 2/ o C AT 4/52AT 2/ o C Storage Dry Ice Dry Ice AT Shipping

Shipping details

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

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

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

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

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.

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