A.M.J. Wensing, MD, PhD

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, CD4-counts 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

0

20

40

60

80

100

NRTI NNRTI Total

Num

ber o

f mut

ation

s

47 7225

Accumulation of drug resistance observed during retrospective testing

baselineT1T2 + 36%

+ 15%+ 76%

44 54 98

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 RT-

PCR 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 TotalNumber of patients56 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, yr4,2 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

median amount of mutations per year of treament

0

1

2

3

4

5

6

0-1 yr 1-2 yr 2-3 yr 3-4 yr 4-5 yr >5yr

= 5 18 911 812

Median number of mutations per year of treatment

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