Implementation of resistance testing on
dried blood spots enables individual patient
management in rural South-African Setting
A.M.J. Wensing, MD, PhD
Introduction
• Antiretroviral programmes have delivered HAART to
6,6 million patients in resource-limited settings
• Monitoring is conducted by clinical symptoms, CD4counts and, if available, viral load
• Overall, effective viral suppression is achieved1
resulting in health improvement and extension of life
1. Barth et al. Lancet Infectious Diseases 2010
Ndlovu Medical Center
• ARV programme initiated in 2003:
First-line ART: NNRTI, 3TC with (d4T) or AZT
• Virological monitoring every 6 months, yearly once
viral suppression has been achieved
• Viral suppression rate (<50 cp/ml) is ca 80%
• After initial virological suppression ca 20% of patients
experience a viral rebound (> 1000 cp/mL)
• Upon repeated testing: resupression or switch
• Ongoing viraemia during first-line ART has been
documented in a limited group of patients
Accumulation of drug resistance observed
during retrospective testing
Number of mutations
100
80
baseline
T1
T2
+ 36%
+ 15%
60
+ 76%
40
20
25
44
47
54
72
98
0
NRTI
NNRTI
Total
Barth et al. Antiviral Therapy in press
Obstacles to resistance testing in RLS
• In case of therapy failure resistance testing is not
generally available in remote settings due to:
•
•
•
•
•
•
•
•
High overall costs
Expensive equipment and limited after sales support
Use of plasma, which requires cold-chain maintenance
Insufficient space for appropriate laboratory flow to prevent
contamination
Unreliable power supply can interrupt procedures, damage
equipment and compromise sample integrity
Unreliable reagent supply
Shortage of skilled laboratory workers
Limited access to expertise for interpretation of results
Pilot Project
• Pilot-project implementing resistance testing on dried
blood spots (DBS) as routine procedure
• Eligibility: patients experiencing viral rebound after initial
viral suppression
• DBS are prepared locally from whole blood.
• Once dried DBS are no longer infectious they can be
shipped at room temperature by airmail to a reference
laboratory.
Test and report procedure
• Inexpensive in house assay was modified for use of DBS
• Nucleic acid is eluted from two 50ul spots using Nuclisens
lysisbuffer
• Nucleic acid is extracted using Minimag, amplified by nested RTPCR for PR-RT and sequenced
• Individual patient reports
• Include susceptibility ranking based on freely available algorithms
• Expert advice is added by a clinical virologist based on observed
resistance patterns, treatment history and local drug availability
• Final report is provided by email within 3 weeks
Email Report
Patient Characteristics
1st line
2nd line
Total
Number of patients
56
28
84
Age, median
36
34
36
Female gender
67%
71%
68%
Pre-therapy CD4, median
74
89
76
CD4 at DBS time
215
224
218
Log HIV-RNA at DBS time
4,2
4,2
4,2
Time on HAART, yr
4,2
3,4
4,0
Time on PI, yr
-
1,7
DBS succes rate
96%
93%
95%
DBS succes rate
100%
100%
100%
when VL >400
Resistance Patterns observed in DBS
%
Extent of resistance was not related to
therapy duration
mediannumber
amountof
ofmutations
mutations per
per year
year of
Median
of treament
treatment
6
5
4
3
2
1
0
0-1 yr
=5
1-2 yr
18
2-3 yr
12
3-4 yr
11
4-5 yr
>5yr
8
9
In Summary
• Resistance testing on DBS was successfully
implemented as a routine clinical procedure in a
rural setting
• Resistance testing using in house methods
enables optimized genotyping of DBS and
reduces processing costs
• Use of DBS sampling enables access to
resistance testing even in rural settings
Acknowledgements: Collaborations
• Ndlovu Medical Center
• Hugo Tempelman
• Peter Schrooders
• Mariette Slabbert
ARTA
University Medical Center Utrecht
•
Andy Hoepelman
www.umcutrecht.nl/virusdiagnostics
UMC Utrecht, Antiviral Escape Group