Advances of Basic Sciences - Diagnostic & Prognostic Application
Advances of Basic Sciences - Diagnostic & Prognostic Application
Advances of Basic Sciences - Diagnostic & Prognostic Application
HHRF 2011
Time of detection of specific markers of HIV infection, according
standardised, commercially available kits.
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Serologic Tests
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Serologic testing algorithms for recent HIV
seroconversion(STARSH)
Monitoring transmission patterns and guiding the interventions
to curb spread
BED, Avidity, IDE-V3, Detuned assays
Accuracy of STARSH
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Serologic testing algorithms for recent HIV
seroconversion(STARSH)
BED: a commercial enzyme immunoassay (EIA) anti-HIV IgG
present in a specimen relative to the total amount of IgG, which is
an indicator of disease progression and provides an estimate of the
time period since seroconversion.
Uses synthetic oligopeptide derived from immunodominant region
of gp41 of HIV-1 subtypes B, CRF_01AE and D
Avidity: modified ELISAs designed to investigate the maturity of
the anti-HIV antibody response by using a chaotropic agent
IDE-V3 : uses two highly conserved immunogenic epitopes of Env
Glycoprotein
Detuned assays: involve increasing the dilution of a known anti-
HIV antibody-positive sample and reducing the incubation time to
deliberately lower the sensitivity of an EIA.
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Accuracy of STARSH
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Challenges for diagnosis
Patient
Treatment Advanced
monitoring Newer
Adherence diagnostics
antiretroviral
Improved s
patient
outcomes
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Monitoring Tests
Lymphocyte analysis (CD4 percentage)
Viral load assays
• Reverse transcription-polymerase chain reaction (RT-PCR) (Roche Amplicor HIV-
1 Monitor and Roche Amplicor HIV-1 Monitor Ultrasensitive)
• Branched chain DNA (bDNA) (Versant HIV-1 RNA 3.0 assay)
• Nucleic acid sequence-based assays (NucliSens HIV-1 QT assay [bioMérieux])
Drug resistance tests
• Genotypic assays
– INNO-LiPA HIV-1 RT (Innogenetic/Bayer Diagnostics)
– Trugene HIV-1 genotyping test (Visible Genetics)
– ViroSeq HIV-1 genotyping system (Applied Biosystems)
• Phenotypic assays
– Antivirogram (Tibotec-Vicro)
– PhenoSense (ViroLogics
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CD4 and viral load estimations
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CD4 counting assays currently undergoing
evaluation, or already in use
Manual bead based technologies
Dynal Manual Dynabeads Dynal® T4 Quant Kit
Beckman Coulter cytosphere kit (Coulter® Manual CD4 Count Kit1-4)
Flow Cytometry systems that require minimal technical input
Guava Easy Cd4223
BD FACSCount
• Point Care (no peer reviewed publications)
• CD4 Select (uses same equipment as machinery for complete blood counts)224
Flow Cytometry based systems that require operator input/flow cytometry skill
• Beckman Coluter flow cytometry(FL, USA) (FlowCare, PLG CD4)
• Becton Dickinson, (Multiset, SP Tetraone)
• Partec (CyFlow™, CyFlow SL Blue , CyFlow Counter®/ CyFLOW(Green)
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Limitations of existing CD4 testing systems
High capital and running costs
unreliable electricity supply, lack of staff with sufficient education to
receive training, distance of health facilities from laboratories and lack of
reliable transport for materials
• A microchip-based assay: currently under development by LabNow.
• Zyomyx: quantitative biochip-based assay due for external validation early in
2009.
• Inverness/ClonDiag assay, uses a CCD imager to image CD4 cells isolated
in an array.
• BeckmanCoulter: semi-quantitative assay, three potential technology
strategies currently under investigation.
• Burnet Institute / Duke and Rush University: semi-quantitative lateral flow
device, final prototype due to enter clinical studies in early 2009.
• Cornell University: liposomal-based lateral flow assay; dye-carrying
liposomes that are stable at high temperatures will bind to CD4 receptors
within the assay..
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A Microchip CD4 Counting Method for HIV
Monitoring in Resource-Poor Settings
Rodriguez WR et al. A microchip CD4 counting method for HIV monitoring in resource-poor settings. PloS Medicine 2: e182,
2005.
POC CD4+ T-lymphocyte test developed
by PATH
Minimally instrumented CD4+ purification technologies
and proprietary flow-through cassettes to measure cell
count status.
Semiquantitatively assesses CD4+ lymphocyte cell
numbers as insufficient (0–250), borderline (250–350),
and sufficient (350 and above) using colorimetric
changes in a flow-through membrane.
Completed in less than two hours and can be performed
by laboratory technicians with low levels of training in
developing countries.
Next steps for this test include fabrication of a capillary
tube collection device, a card-based purification system,
and further optimization of the membrane-based signal
system.
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POC for CD4 counts
Low sensitivity;
Unable to quantify moderate changes in CD4 count indicative of
treatment failure,
Need to determine what sort of assays will be required in order to
support treatment switch decisions.
Further questions:
How sensitive do point of care CD4 assays need to be? Are they
only needed for staging individuals for treatment initiation, or for
monitoring for treatment failure too?
What is the relative need for point of care versus more sensitive
assays likely to be in the medium term
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Viral load estimation
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Current FDA-licensed assays
Fiscus SA, Cheng B, Crowe SA. HIV-1 viral load assays for resource-limited settings. PLoS Medicine 3 (10): e107, 2006.
Greengrass V., et al. Evaluation of the Cavidi ExaVir™ Load quantitative HIV RT load kit as an alternative HIV viral load monitoring
test for use in Melbourne. Fourth International AIDS Society Conference on HIV Treatment and Pathogenesis, Sydney, abstract
TUPEB019, 2007.
An ideal viral load test
No more than a fingerstick’s worth of blood (about a tenth of a millilitre),
a single cartridge into which a sample of blood could be inserted and
which would give a result, that it should not require refrigeration, be able
to be run on batteries,
should not cost more than $1000 per instrument and $8 per test, should
give a result within two hours
would be able to be done by a field health worker with 1-2 days’ training.
Approaches to delivering a viable alternative include:
SAMBA – nucleic acid-based test kit being developed by the University
of Cambridge and Diagnostics for the Real World. Being field tested in
Kenya
Siemens (now owner of Bayer): battery operated, hand-held nucleic
acid-based testing device, being developed in collaboration with IAVI
and NIAID.
University of Maryland: anti-p24 antibody capture with an amplification
step to improve on current ELISA-based p24 tests.
Product concept
Sample processing device
Patient Specimen RNA Specimen
collection extraction preservation
Cold chain
Diagnostic Test
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PATH sample processing technology
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Path sample processing technology
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Monitoring Drug resistance
Genotyping assays
Ultra Deep Sequencing technologies
Phenotyping assays: DR and Tropism
Virtual phenotyping
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Genotypic Assays
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Cell based Phenotypic assays
Determine dug resistance and tropism
Patient’s viral reverse-transcription and protease genes
incorporated into a laboratory virus strain grown in the presence of
varying concentrations of ARVs.
Viral susceptibility are presented in the context of either biological
cutoffs or clinical cutoffs
More useful for complex mutation patterns where genotypic
interpretation may be difficult.
Phenotypic assays cannot specifically account for the combined
activity or lack of antiretroviral combinations and less predicitive
when mixed strains present
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Virtual Phenotype
Winters B, Montaner J, Harrigan PR, Gazzard B, Pozniak A, Miller MD, Emery S, van Leth F, Robinson P,
Baxter JD, Perez-Elias M, Castor D, Hammer S, Rinehart A, Vermeiren H, Van Craenenbroeck E, Bacheler L. J
Acquir Immune Defic Syndr. 2008 May 1;48(1):26-34.
Mazzotta F, Lo Caputo S, Torti C, Tinelli C, Pierotti P, Castelli F, Lazzarin A, Angarano G, Maserati R, Gianotti
N, Ladisa N, Quiros-Roldan E, Rinehart AR, Carosi G. J Acquir Immune Defic Syndr. 2003 Mar 1;32(3):268-80.
Hales G, Birch C, Crowe S, Workman C, Hoy JF, Law MG, Kelleher AD, Lincoln D, Emery S.PLoS Clin Trials.
2006 Jul 28;1(3):e18
Ultra deep sequencing
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Resistance testing : a cornerstone of
antiretroviral therapy selection
Improving patient outcomes:
Selection of the appropriate resistance test is based on the patient’s clinical
history and drug exposure
Early use of tropism assays may select patients who are candidates for
CCR5 entry inhibitors before virus populations change to mixed tropism
Strengthening surveillance
Ultra-deep sequencing technologies have the ability to detect drug-resistant
and low-frequency HIV strains.
The WHO is leading an initiative to genotype and monitor drug resistance
in RLS
Develop genotype panels, data bases testing protocols and research
agendas
60 nations adopted this initiative hence presents an opportunity for
capturing data and minimize DR
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Challenges