CD4 and Viral Load Diagnostics for the Developing World

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CD4 and Viral Load Diagnostics for the Developing
World

• Elizabeth M. Dax, and the staff of the NRL,
  Australia

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Talk will cover-

• Introduction the need for Cost-Effective
  approaches
• Methods available
• Viral Load - Disadvantages and advantages
• CD4 - Disadvantages and advantages
• Approaches that will assist
• What can be done e.g. TAQAS

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Introduction

• Estimated 40 million living with HIV
• Most do not have obvious risk factors
• International programmes now offer antiretroviral
  therapy e.g.
• GFATM
• PepFAR
• World and AD Banks
• Most do not have access to adequate laboratory
  services but necessary to know who to treat!

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In a population where there are no risks other
than heterosexual transmission if.

• Estimated prevalence x
• Have to test (100-x) times the estimated number
  of infected to identify all infected in that
  population
• Thus, to find those who could be treated may be
  an extensive exercise
• Cost of screening must be included in estimates
• 10 of those infected will require treatment and
  monitoring

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Lower Cost Approaches to Viral Load

• Centralised systems
• DBS (samples shipped to central testing
  laboratory)
• centralised testing laboratories
• Immunoassays
• ExaVir RT EIA
• Quantitative p24 Ag
• Rapid test with limits
• More C-E Conventional techniques
• Real time PCR

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Cavidi ExaVir? Load - an ultrasensitive RT assay
A low-cost, low-tech HIV viral load assay
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ExaVir Load RT assays

• Measures RT activity ? equiv. RNA copies/ml
• Medium throughput (30 tests/run 1 scientist
  ?30-90/wk)
• Affordable
• Less prone to contamination than RT-PCR
• Technically simple, largely manual
• Equipment required is minimal and robust
• Detection range 400 to 1,000,000 copies/ml
• Requires 1ml plasma (but less works OK)
• Detects all HIV-1 subtypes, HIV-2 and SIV
• CE Marked and ISO certified

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Results - Sensitivity

• All specimens gave valid results
• Detectable RT activity was present in 91 of
  samples with HIV RNA gt1,000 copies/mL
• Detectable RT activity was present in 99 of
  samples with HIV RNA gt10,000 copies/mL
• All but one cultured isolate gave a positive
  result

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Sensitivity Viral Loads
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ExaVir Load ver 2 vs ver 3

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Review of Newer CD4 Technologies

• Suzanne Crowe
• Burnet Institute for Medical Research and Public
  Health
• Melbourne

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Current CD4 monitoring tests and technologies
include

• Flow based assays
• Becton Dickinson FACSCount
• Guava EasyCD4
• Partec CyFlow
• Beckman Coulter PointCARE
• Manual immune bead-based assays
• Dynal Dynabeads
• Coulter Cytosphere
• In the pipeline
• LabNow microchip technology
• SemiBio slide test (microscopy)

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Comparison of FACSCount with Guava EasyCD4 in
Chennai
100
1200
N 110 R 0.98
1000
0
800
CD4 by FLOW CYTOMETRY
DIFFERENCE
600
-100
400
Bias 26 for EasyCD4
200
0
-200
1200
1000
800
600
400
200
0
1000
800
600
400
200
0
1200
CD4 by EASYCD4
MEAN
Balakrishnan et al, 2005
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CD4 counts by flow cytometry and Coulter
Cytosphere assay
300
n 122
200
100
Difference
0
-100
-200
-300
1200
1000
800
600
400
200
0
Mean
Balakrishnan et al JAIDS 2004 36 1006-1010
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Should low-cost CD4 assays be introduced?
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• Depends on
• Number of samples per day
• Low throughput, manual may be most cost effective
• High throughput, flow method most practical
• Sophistication of lab
• methods require varying degree of technical skill
• Availability of technical support
• A key issue for flow cytometers
• Remote area, opt for
• manual or
• ship samples or
• ensure local engineers trained
• Cost

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Where are we up to?

• In-country analyses continuing.
• Rigorous independent evaluation is absolutely
  essential
• Some technologies /assays recently licensed in
  USA (PLG, PointCARE)
• However all are still emerging technologies
• QA participation essential establishing this
  should be part of the deal
• Countries should not purchase technologies that
  have not been adequately validated

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Monitoring Infections Maximises-

• Good management
• Treatment efficaciousness
• Avoiding drug resistance
• Management of toxicities

Capacity Building necessary
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Other Approaches

• Laboratory systems rather than more laboratories

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Laboratory Systems
NRL(s)
Refer Samples
Central (Reference) Labs
Intermediate Labs
Peripheral Labs
QA
Health Care Settings
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TAQAS

• TreatAsia Quality Assessment Scheme for HIV-1
  Genotypic Resistance Testing

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TREATAsia Network and TAQAS

• Evaluation, surveillance and monitoring of HIV
  drug resistance in Asian region.
• Quality Assessment Scheme (TAQAS).
• Detection of drug resistance mutations.
• Interpretation and reporting of genotypic data.
• Identification of technical factors affecting
  optimal performance.
• Standardizes outcomes HIV genotypic resistance
  testing.
• Meaningful and confident comparison of results.

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TAQAS Laboratories
Current Laboratories
Future Laboratories
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Aligning of Nucleotide Sequences
Positions associated with drug resistance
Key
Wildtype
Mixture
Mutant
Differences in nucleotide sequence are made
obvious to laboratories to enable self-education.
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Detection of drug resistance mutations
Most laboratories detected gt85 of the drug
resistance mutations (DRM) in Panel 1. Detection
of DRM was sustained in Panel 2.
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TAQAS Outcomes .to date

• High quality genotyping results achieved in a
  group of Asian laboratories.
• Some differences identified between laboratories
  in the outcomes of
• sequencing
• sequence editing
• detection of drug resistance mutations.
• The scheme will be expanded and the variation in
  outcomes of HIV genotyping between laboratories
  addressed.

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New Approaches

• Laboratory systems rather than more laboratories
• Transport systems

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New Approaches

• Laboratory systems rather than more laboratories
• Transport systems
• Better tests for use in resource-limited
  settings

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New Approaches

• Laboratory systems rather than more laboratories
• Transport systems
• Better tests for use in resource-limited
  settings
• Training

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New Approaches

• Referral systems rather than more laboratories
• Transport systems
• Better tests for use in resource-limited
  settings
• Training
• Improved laboratory development mechanisms
  including QA

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New Approaches

• Laboratory systems rather than more laboratories
• Transport systems
• Better tests for use in resource-limited
  settings
• Training
• Improved laboratory development mechanisms
  including QA
• Insist on donors supporting systems and
  infrastructure - not individuals

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• Laboratories are once again are seen as important
  
• Screening
• Monitoring

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Summary

• There is urgent need for Cost-Effective
  approaches
• Newer methods are available for Viral Load and
  CD4 but each has its own advantages and
  disadvantages
• There are some approaches that must be considered

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Acknowledgements

• The Forum CD4 working group members, especially
• Veronica Miller
• Ben Cheng
• Susan Fiscus
• Alan Landay
• Bill Rodriguez
• Clinical Research lab at the Burnet Institute,
  especially
• Vicki Greengrass, Lisa Morris
• Others who contributed new slides or information
  for this presentation
• Roland Gohde (Partec)
• Dr Balakrishnan (YRG Care Chennai)
• Frank Xu (Semi-Bio)
• John Wotherspoon (Becton Dickinson)
• Jeff Harvey, Tina Baumgartner, Leonard Buchner,
  (Guava),
• AngieVernon (Beckman Coulter, PointCare),
• Viv Granger and Dave Barnett (NEQAS), Amanda
  Lindholm (Streck),
• Shelley Hossenlopp and Larry Fox (LabNow)