Susan A. Fiscus, Ph.D.
University of North Carolina at
Chapel Hill

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

• 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

 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

 “Cheap, fast, or accurate. Pick 2” (Dr.
Bill Rodriguez, Harvard Univ, Nov 16,
2009)

• 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

• 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.

• 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 HIV-
1 RNA Assays
Manufacturer
Gen-Probe
Assay Name
Aptima
Target
LTR and pol
HIV-1 Subgroup
Recognition
Group M: A-H
Group N and O
Range (RNA
Copies/mL)
Qualitative
Roche Amplicor HIV Monitor v1.5 Gag
Group M: A-H
(underquantitates some subtypes)
Standard: 400 to 750,000
Ultrasensitive:
50 to 100,000
Roche
COBAS
AmpliPrep/COBAS
TaqMan HIV-1 Test, version 1.0, 2.0
Gag
Group M: A-H
(v. 1. may under-quantitate some subtypes; improved with v.2)
20-40 to 10,000,000
Siemans
Abbott bioMerieux
Biocentric
Cavidi
Perkin Elmer Life
Sciences
Versant HIV-1 RNA 3.0
(bDNA)
RealTime HIV-1 Assay
NucliSENS EasyQ HIV-1 v2.0 (RUO US)
Generic HIV Charge Virale
Cavidi ExaVir v.3
Ultrasensitive p24 Ag
Assay
Pol
Int
Gag
LTR
RT p24
Group M: A-H
Group M: A-H
Group O, N, P
Group M: A-H
Group M: A-H
Group N
Group M: A-H, Group O, N
HIV-2
Group M Subtypes: A, B, C,
E, AE, AG
U.S.: 75-500,000
Ex U.S.:
50-500,000
40 to 10,000,000
25 to 10,000,000 cps/mL
300 to 5,000,000
~200 to 600,000 cp equivalents/mL
Difficult to determine from package insert

• 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

• 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

3
2
1
0
0
7
6 y = 0.9817x + 0.1187
R
2
= 0.9157
5
4
1 2 3 4
Reference Assay VL(log10)
5
 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
6 7
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)
5
IMI negative
73 (28 %)
IMI positive
56 (22 %)
4
3
COBAS plasma negative
COBAS plasma
<40 cp/ml
COBAS plasma positive
8 (3 %)
6 (2 %)
21( 8 %)
94 (36%)
2
1
0
0 1 2 3 log cp/ml COBAS pE
4 5 6
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 HIVnegative donors) donors receiving HAART therapy naïve donors
——— blood viral load equals plasma viral load

Lee, et al 2010. JID 201 Suppl 1:S65-72

Comparison of the Simple Amplification-Based Assay (SAMBA) HIV-1
Test with Commercially Available HIV-1 Nucleic Acid Tests
Feature Abbott
RealTime
<10 C o
NucliSens Roche
COBAS
2-8 C o 2-8 C o
SAMBA HIV-1
Cold storage
Sample volume
200 uL 1.0 mL 200 uL
Not required
240 uL
Sensitivity 150 cp/mL
Subtypes
176 cp/mL
M: A-H, N, O, P M: A-H
400 cp/mL
M: A-H
200 cp/mL
M: A-K, N, O
From Lee, et al 2010. JID 201 Suppl 1:S65-72

• 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

• 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

• 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: 25 m
L
Immune Disruption: 90 o C heat shock
Assay Steps:
1. Add 25 m
L plasma to 75 m
L 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 98-
100%)
• 5 samples gelled (1.3%) giving invalid results

Point-Of-Care p24 Antigen Rapid Test
Under Development
Assay Procedure
1. Separate plasma
2. Pretreat sample in processor
Whole blood volume: 80 m
L
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?
3. Insert rapid test strip and read results

Cheap:
(< $ 5 USD)
Fast:
< 60 min
Accurate:
Sensitivity: > 95%
Specificity: > 98%
IQuum
Inverness
SAMBA
CIGHT p24
??????
??????
??????
< 2 hrs
???????

• 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 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

• 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 - ?????

 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