Transcript Field Study of the Utility of Dried Blood Spots (DBS) for

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 -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 AT 4/52 AT 2/52
-80oC
-80oC
Storage
Dry
Ice
Dry
Ice
AT
AT
Shipping
AT
AT
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