1. EID - Current tools and prospects of point of care technology
Susan A. Fiscus, Ph.D.
University of North Carolina at Chapel Hill

2. Disclosures Honoraria: Gen-Probe, Roche, Abbott
Free kits: Roche, Gen-Probe, Perkin-Elmer, Cavidi, Siemans, Abbott, Inverness, IQuum, ImmunoDiagnostics

3. 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

4. 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

5. Desirable Qualities of a POC Test
“Cheap, fast, or accurate. Pick 2” (Dr. Bill Rodriguez, Harvard Univ, Nov 16, 2009)

6. 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

7. 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.

8. 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

9. Commercially Available FDA Cleared HIV-1 RNA Assays
Manufacturer
Assay Name
Target
HIV-1 Subgroup Recognition
Range (RNA Copies/mL)
Gen-Probe
Aptima
LTR and pol
Group M: A-H
Group N and O
Qualitative
Roche
Amplicor HIV Monitor v1.5
Gag
(underquantitates some subtypes)
Standard: 400 to 750,000
Ultrasensitive:
50 to 100,000
COBAS
AmpliPrep/COBAS
TaqMan HIV-1 Test,
version 1.0, 2.0
(v. 1. may under-quantitate some subtypes;
improved with v.2)
20-40 to 10,000,000
Siemans
Versant HIV-1 RNA 3.0 (bDNA)
Pol
U.S.: ,000
Ex U.S.:
50-500,000
Abbott
RealTime HIV-1 Assay
Int
Group O, N, P
40 to 10,000,000
bioMerieux
NucliSENS EasyQ HIV-1 v2.0 (RUO US)
25 to 10,000,000
cps/mL
Biocentric
Generic HIV Charge Virale
LTR
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
Perkin Elmer Life Sciences
Ultrasensitive p24 Ag Assay
p24
Group M Subtypes: A, B, C, E, AE, AG
Difficult to determine from package insert

10. 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

11. 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 % correlation coefficient
Siemens Versant – 92% correlation coefficient
Haven’t tested whole blood yet. Limited experience with clinical samples. Can operate on a battery.

12. 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
Fiscus, unpublished data 2010

13. 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

14. 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
positive
COBAS
plasma
73 (28 %)
56 (22 %)
<40 cp/ml
8 (3 %)
21( 8 %)
6 (2 %)
94 (36%)
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
specificity of both assays =100% (32 HIV-negative donors)
258 clinical samples from HIV infected individuals of unknown subtypes were tested in both the Clondiag POC test using 10 ul whole blood and Roche Taqman using 1.0 ml of plasma. I’m not sure whether this was version 1.0 or 2.0 of Taqman.
donors receiving HAART
therapy naïve donors
blood viral load equals plasma viral load
———

15. SAMBA HIV-1 POC Test
Lee, et al JID 201 Suppl 1:S65-72

16. 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
Not required
Sample volume
200 uL
1.0 mL
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-K, N, O
From Lee, et al JID 201 Suppl 1:S65-72

17. 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

18. HIV-1 p24 Antigen Tests
The ultrasensitive, heat dissociated p24 antigen assay has been shown to work well for EID
With both plasma
sensitivity %
specificity %
N= 2314 samples from 9 publications
And DBS
Sensitivity – %
Specificity – 100%
N=1328 from 3 publications

19. Point of Care p24 Antigen Tests
Inverness Determine Combination Ab/p24 Ag
ImmunoDiagnostics
mBio Diagnostics
Northwestern –Abbott partnership - David Kelso

20. 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 20

21. 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 %)
5 samples gelled (1.3%) giving invalid results 21

22. 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.
Consumables: Plasma separator, reaction tube, reaction buffer, rapid test strip
Processor: Battery operated
Cost per Assay: $1-2 per test
Ready early 2012?
2. Pretreat sample in processor
3. Insert rapid test strip and read results 22

23. “Cheap, fast, or accurate. Pick 2”
(< $ 5 USD)
Fast:
< 60 min
Accurate:
Sensitivity: > 95%
Specificity: > 98%
IQuum
??????
Inverness
???????
SAMBA
< 2 hrs
CIGHT p24

24. 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

25. 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 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

26. 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 - ?????

27. 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