Field Study of the Utility of Dried Blood Spots (DBS) for HIV-1 Drug Resistance (HIVDR) Genotyping
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Transcript of Field Study of the Utility of Dried Blood Spots (DBS) for HIV-1 Drug Resistance (HIVDR) Genotyping
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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
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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)
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WHO HIVDR Laboratories
This map is an approximation of actual country bordersDecember 2011
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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)
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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
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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%)
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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
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Shipping details
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Effect of Temp During Shipment
* p=0.003 vs Shipment 2Only 4 shipments with temp data and 10 or more samples included in analysis
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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
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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
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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
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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.
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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