Early Infant Diagnosis:
Current Tools and Prospects of
Point of Care Technology
Susan A. Fiscus, Ph.D.
University of North Carolina at
Chapel Hill
Disclosures
• Honoraria: Gen-Probe, Roche, Abbott
• Free kits: Roche, Gen-Probe, PerkinElmer, Cavidi, Siemans, Abbott,
Inverness, IQuum, ImmunoDiagnostics
Acknowledgments
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Dr. Shuqi Chen, IQuum
Dr. Robert Coombs, Univ of Washington
Dr. John Gerdes, Micronics
Dr. Jeanne Jordan, George Washington Univ
Dr. David Kelso, Northwestern Univ
Dr. Helen Lee, Univ of Cambridge
Dr. Laura Mazzola, WAVE 80
Dr. Christopher Myatt, mBio Diagnostics
Dr. Avi Pelossof, Inverness
Dr. Michael Pollack, Advanced Liquid Logic
Desirable Qualities of a POC
Diagnostic Test
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


Rapid (< 1 hour)
Sensitive (how sensitive? > 95%)
Specific (how specific? > 98%)
Inexpensive (< $5/test)
Simple
 Equipment – battery operated, few moving parts,
small footprint
 Technique – minimal training required
 Robust - No cold chain requirement
 Commercially available
 CE marked/FDA cleared
Desirable Qualities of a POC
Diagnostic Test
 “Cheap, fast, or accurate. Pick 2”
(Dr. Bill Rodriguez, Harvard Univ, Nov 16,
2009)
HIV DNA and Total Nucleic Acid Assays
• Roche AMPLICOR HIV DNA assay, v 1.5 is the
gold standard
– Has been successfully introduced and implemented
in many countries around the world
– Can use whole blood pellets or Dried Blood Spots
(DBS)
• Roche Qualitative Total Nucleic Acid Assay has
been introduced (Stevens, et al, JCM 2008)
– Works on whole blood and DBS
– 100% sensitive and 99.7% specific
• Abbott also developing a DNA assay
• Both Roche and Abbott assays require large,
expensive new equipment
– Probably suitable for large centralized labs
POC HIV DNA Assays
• CIGHT, Dr D Kelso, Northwestern Univ
– LoD 5 cp/reaction (Jangam, 2010, CROI)
– not yet ready for field testing and on hold while
work focuses on a POC p24 test
• Micronics – Real Time PCR (Tim Granade,
CDC; CROI 2010)
• BioHelix – isothermal lateral flow – 2 hr TAT
(Jeanne Jordan, GWU; HIV Diagnostics Mtg,
2010)
• Both Micronics and BioHelix seem to be
more in the proof of concept stage and don’t
yet seem ready for field testing.
HIV RNA Assays
• Qualitative Gen-Probe Aptima
– Only HIV RNA assay FDA approved for diagnosis
(though approval is for plasma or serum, not DBS)
– Works well with DBS
– Very sensitive and specific (Kerr, 2009; Stevens, 2009)
– Being used by the State of New York for EID
• Quantitative HIV RNA assays may not be as
sensitive when infants are being prophylaxed or
if mothers are receiving ARVs and the child is
breast-feeding
Commercially Available HIV-1 Viral Load
Assays
Manufacturer
Assay Name
Roche
Amplicor HIV Monitor v1.5 , COBAS
Roche
TaqMan HIV-1, version 1.0, 2.0
Siemans
Versant HIV-1 RNA 3.0 (bDNA)
Abbott
bioMerieux
RealTime HIV-1 Assay
NucliSENS EasyQ HIV-1 v2.0 (RUO US)
Biocentric
Generic HIV Charge Virale
Cavidi
Cavidi ExaVir v.3
POC RNA Assays
Univ of Cambridge & Diagnostics for the Real
World – isothermal amplification with visual
detection by dipstick, LoD 75 cp/mL using
250uL plasma, <90 min
IQuum – realtime PCR, LoD – ~100 cp/mL, 1
hr, 200 uL plasma
Inverness – microarray, realtime detection,10
uL whole blood
• Advanced Liquid Logic - based on digital
microfluidics
• Wave 80 – assay based on bDNA assay
SAMBA HIV-1 POC Test
Lee, et al 2010. JID 201 Suppl 1:S65-72
SAMBA
• Semi-quantitative VL assay with cut-off of 1,000 cp/mL
• Qualitative assay appropriate for EID
• 250 uL plasma - limit of detection 75 cp/mL
100 uL whole blood - limit of detection 400 cp/mL
• Robust – no cold chain required, can be battery
operated
• Simplified sample preparation chemistry
• Sample prep and amplification/detection equipment
not linked currently
• Little training required
• Field testing in resource-limited settings in September
2010
• Clinical trial for regulatory approval in 2011
LIAT™ Quantitative POC HIV Assay
• 200 uL plasma sample input (haven’t tested whole
blood yet)
• Realtime PCR
• Each cartridge has an internal control
• Dynamic range 100 to 10 million cp/mL in 60 min
• Detects HIV-1 Groups M and O and HIV-2 viruses
•Can be battery operated
•Very simple training
and operation
•Add 200 uL plasma to
cartridge, cap, and
insert in instrument
LIAT
92% correlation
with Abbott m2000
with 75 clinical
specimens (clades
A, B, C, and D)
Training took 5
minutes
Easy to use
Assay takes 60
minutes
7
y = 0.9817x + 0.1187
R2 = 0.9157
6
Liat Assay VL (log10)
5
4
3
2
1
0
0
1
2
3
4
5
6
7
Reference Assay VL(log10)
Fiscus, unpublished data 2010
IMI’s CLONDIAG HIV Viral Load
Point-of-Care Test
Can use fingerstick, whole
blood, or plasma.
Multiple HIV-1 and HIV-2 targets
are detected simultaneously by a
proprietary microarray real time
detection method.
The test includes internal
controls
The sample is applied directly
onto the test cartridge
The cartridge is processed by a
compact, battery driven
instrument
IMI CLONDIAG HIV Viral Load Test
1 ml of EDTA Plasma (COBAS Ampliprep/Taqman) versus
10 µl of Whole Blood (IMI’s prototype assay)
log cp/10 µl IMI wbE
5
4
Percentage of samples with
detectable viral load:
3
COBAS (1 ml plasma) 50 %
IMI VL (10 µl blood) 66 %
all samples are from HIVpositive donors
2
specificity of both assays
=100% (32 HIV-negative donors)
1
0
0
N=258
1
2
3
4
5
log cp/ml COBAS pE
donors receiving HAART
therapy naïve donors
——— blood viral load equals plasma viral load
6
HIV-1 p24 Antigen Tests
• The ultrasensitive, heat dissociated p24
antigen assay has been shown to work
well for EID
– With both plasma
• sensitivity - 91-100%
• specificity - 95-100%
• N= 2314 samples from 9 publications
– And DBS
• Sensitivity – 98-100%
• Specificity – 100%
• N=1328 from 3 publications
Point of Care p24 Antigen Tests
•
•
•
•
Inverness Determine Combination Ab/p24 Ag
ImmunoDiagnostics
mBio Diagnostics
Northwestern –Abbott partnership - David
Kelso
p24 Antigen Rapid Test for
Diagnosis of Acute Pediatric HIV Infection
Assay Steps:
1. Add 25mL plasma to 75mL buffer
2. Heat in water bath at 90oC for 4 min
3. Insert test strip & read after 20 min.
Assay Sensitivity:
50pg/mL or 40,000 RNA copies/mL
p24 Rapid Test Strip
Results from pre-clinical trials in Cape Town
• 394 infant samples tested at NHLS Virology Lab,
Groote Schuur Hospital, Cape Town, South Africa
• 86% of samples were from babies < 6 months of age
• 53% from infants < 2 months of age
• Reference Assay: Total Nucleic Acid PCR (Roche
Ampliprep/COBAS Taqman HIV-1)
• p24 Assay Sensitivity: 23/24 = 95.8% (95% CI 80-99%)
• p24 Assay Specificity: 363/365 = 99.4% (95% CI 98100%)
• 5 samples gelled (1.3%) giving invalid results
Point-Of-Care p24 Antigen Rapid Test
Under Development
Assay Procedure
1. Separate
plasma
Whole blood volume: 80mL
Immune Disruption: Heat shock
Total Assay Duration: 35 min.
2. Pretreat sample in
processor
Consumables: Plasma
separator, reaction tube,
reaction buffer, rapid test strip
Processor: Battery operated
Cost per Assay: $1-2 per test
Ready early 2012?
3. Insert rapid test
strip and read results
“Cheap, fast, or accurate. Pick 2”
Cheap:
(< $ 5
USD)
IQuum
?????
Inverness
?????
SAMBA
CIGHT
p24
$1020?
Fast:
< 60
min
Accurate:
Sensitivity:
> 95%
Specificity:
> 98%
?????
< 90
min
Whole
blood
Robust
(battery
operated and
no cold
chain)
In
development
Needs cold
chain
Centralized vs POC Testing
Centralized Testing using
DBS
• Can be implemented now
• Better control on training,
supply logistics, internal
and external QA
• High through-put
----------------------• Huge backlog of DBS in
some countries with long
turn around times
• Delays and problems in
returning results
Point of Care
• Results ready in an hr or
less
• Possibly fewer problems
with mislabeling
• Able to confirm positive
test results immediately
-------------------• Potential problems with
training, competency,
logistics
• Not yet ready for prime
time
Key Points
• POC assays should be inexpensive,
rapid, simple, sensitive, specific, and
robust
• Promising POC assays today include:
IQuum’s LIAT, SAMBA, CIGHT’s p24,
and possibly Clondiag’s VL assay
Steps to move forward
 Continue lab validation of new POC tests
 Field test new assays under controlled conditions
 Expand usage and evaluate the effects of POC on
key operational parameters:
 % of infants tested
 % of infants who receive their results
 % of infected infants who access care
 % of infected infants who die or are
hospitalized before age 2 years
 Continuous QA of POC facilities
 Confirmation of all positives at a
central/reference lab