Transcript Serological and Virological Tests to measure PMTCT Impact

EID - 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
Outline
• Desirable qualities for POC assays
• Assays used for EID
– HIV DNA
– HIV RNA
– P24 Antigen
• Considerations when selecting an EID
assay
• Key points
• Steps to move forward
Desirable Qualities of a POC Test
 Inexpensive (< $USD 5 /test)
 Rapid (< 1 hour)
 Simple
 Equipment – battery operated, few moving parts,
small footprint
 Technique – minimal training required
 Sensitive (how sensitive? > 95%?)
 Specific (how specific? > 98%?)
 Robust - No cold chain requirement
 Commercially available/CE marked-FRA cleared
Desirable Qualities of a POC Test
 “Cheap, fast, or accurate. Pick 2” (Dr.
Bill Rodriguez, Harvard Univ, Nov 16,
2009)
HIV DNA Assays
• Roche AMPLICOR HIV DNA assay, v 1.5
is the gold standard – either using
whole blood pellets or Dried Blood
Spots (DBS)
• Has been successfully introduced and
implemented in many countries around
the world
POC HIV DNA Assays
• CIGHT, 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)
• 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
• Have been used as alternatives to HIV DNA
testing
• 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
• Qualitative Gen-Probe Aptima
– Very sensitive and specific (Kerr, 2009; Stevens, 2009)
– Works well with DBS
– Only HIV RNA assay FDA approved for diagnosis
(though approval is for plasma or serum, not DBS)
– Being used by the State of New York for EID
Commercially Available FDA Cleared HIV1 RNA Assays
Manufacturer
Assay Name
Gen-Probe
Aptima
Roche
Amplicor HIV Monitor v1.5
Target
LTR and pol
Gag
HIV-1 Subgroup
Recognition
Group M: A-H
Group N and O
Group M: A-H
(underquantitates some
subtypes)
Qualitative
Standard: 400 to 750,000
Ultrasensitive:
50 to 100,000
Roche
COBAS
AmpliPrep/COBAS
TaqMan HIV-1 Test,
version 1.0, 2.0
Siemans
Versant HIV-1 RNA 3.0
(bDNA)
Pol
Group M: A-H
Abbott
RealTime HIV-1 Assay
Int
Group M: A-H
Group O, N, P
bioMerieux
NucliSENS EasyQ HIV-1
v2.0 (RUO US)
Gag
Biocentric
Generic HIV Charge Virale
LTR
Group M: A-H
Group N
300 to 5,000,000
Cavidi
Cavidi ExaVir v.3
RT
Group M: A-H, Group O, N
HIV-2
~200 to 600,000 cp
equivalents/mL
Ultrasensitive p24 Ag
Assay
p24
Group M Subtypes: A, B, C,
E, AE, AG
Gag
Group M: A-H
(v. 1. may under-quantitate
some subtypes;
improved with v.2)
Range (RNA
Copies/mL)
20-40 to 10,000,000
U.S.: 75-500,000
Perkin Elmer Life
Sciences
Group M: A-H
Ex U.S.:
50-500,000
40 to 10,000,000
25 to 10,000,000
cps/mL
Difficult to determine from
package insert
POC RNA Assays
• IQuum – realtime PCR, LoD – ~100
cp/mL, 1 hr, 200 uL plasma
• Inverness – microarray, realtime
detection,10 uL whole blood
• Helen Lee – semiquantitative dipstick
with 200 cp/mL cutoff (Lee, JID 2010)
• Advanced Liquid Logic - based on
digital microfluidics
• Wave 80 – assay based on bDNA assay
LIAT™ Quantitative POC HIV Assay
• 200 uL plasma sample input (haven’t tested whole
blood yet)
• Limit of detection - 78 copies/mL of ARNA detected
in 60 min
• 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
• Comparative data with 30 clinical specimens:
– Roche COBAS - 88.4% correlation coefficient
– Siemens Versant – 92% correlation coefficient
LIAT
92% correlation
with Abbott m2000
with 75 clinical
specimens (clades
A, B, C, and D)
Training took 10
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
Allows determination of HIV
load in 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 VL Test
Data generated on 1 ml of EDTA Plasma (COBAS Ampliprep/Taqman) versus
10 µl of Whole Blood (IMI’s prototype assay)
IMI
negative
5
COBAS
plasma
negative
COBAS
plasma
<40 cp/ml
COBAS
plasma
positive
log cp/10 µl IMI wbE
4
3
IMI
positive
73 (28 %)
56 (22 %)
8 (3 %)
21( 8 %)
6 (2 %)
94 (36%)
2
Percentage of samples with detectable viral
load:
COBAS (1 ml plasma) 50 %
IMI VL (10 µl blood) 66 %
all samples are from HIV-positive donors
1
0
0
1
2
3
4
log cp/ml COBAS pE
donors receiving HAART
therapy naïve donors
——— blood viral load equals plasma viral load
5
6
specificity of both assays =100% (32 HIVnegative donors)
SAMBA HIV-1 POC Test
Lee, et al 2010. JID 201 Suppl 1:S65-72
SAMBA HIV-1 POC Test
Comparison of the Simple Amplification-Based Assay (SAMBA) HIV-1
Test with Commercially Available HIV-1 Nucleic Acid Tests
Feature
Abbott
RealTime
NucliSens
Roche
COBAS
SAMBA HIV-1
Cold
storage
<10 Co
2-8 Co
2-8 Co
Not required
Sample
volume
200 uL
1.0 mL
200 uL
240 uL
Sensitivity
150 cp/mL
176 cp/mL
400 cp/mL
200 cp/mL
Subtypes
M: A-H, N, O, P M: A-H
M: A-H
M: A-K, N, O
From Lee, et al 2010. JID 201 Suppl 1:S65-72
Total Nucleic Acid Assays
• Roche Taqman - CAP/CTM HIV-1 Qual is
being introduced (Stevens, et al, JCM 2008)
– Works on whole blood and DBS – 100%
sensitive/99.7% specific
• Abbott is developing a total nucleic acid
assay as well
• Both require expensive new instrumentation
– Roche Taqman or Abbott m2000
– Limited information about the performance
of these assays in more resource
constrained settings
– Probably suitable for large centralized labs
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
Plasma volume: 25mL
Immune Disruption: 90oC heat shock
Assay Steps:
1. Add 25mL plasma to 75mL buffer
2. Heat in water bath for 4min
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 infants under 6 months of age,
53% from infants under 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
Fast:
< 60 min
Accurate:
Sensitivity: > 95%
Specificity: > 98%
???????
< 2 hrs
Factors to be Considered When
Selecting an EID Assay
• Performance characteristics
– Sensitivity and specificity
– Specimen type and volume
– HIV subtype(s) in the population
• Technical and support issues
–
–
–
–
Volume and throughput – 1-2 or 1000/day
Equipment footprint
Printable results
Training requirements
• Acceptance by MOH and clinicians
• External and internal quality assurance
Centralized vs POC Testing
Centralized Testing using
DBS
• Can be implemented now
• Better control on training,
supply logistics, internal
and external QA
• Potential for high throughput
----------------------• 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
• Timeline for field testing and implementation:
–
–
–
–
CIGHT p24 – Late 2011-early 2012
IQuum - ????
Inverness - ????
SAMBA - ?????
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