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 ParryMRC/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 bordersDecember 2011

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)

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

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= 0.002 vs group 3

(finger prick)-80oC -80oC -80oC AT 2/52

-80oCAT 4/52 AT 2/52

-80oCStorage

DryIce

DryIce

AT AT AT AT Shipping

Shipping details

Effect of Temp During Shipment

* p=0.003 vs Shipment 2Only 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