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, Perkin-Elmer, 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 HIV-1 RNA Assays

Manufacturer Assay Name TargetHIV-1 Subgroup

RecognitionRange (RNA

Copies/mL)

Gen-Probe Aptima LTR and polGroup M: A-H

Group N and OQualitative

Roche Amplicor HIV Monitor v1.5 GagGroup M: A-H

(underquantitates some subtypes)

Standard: 400 to 750,000

Ultrasensitive: 50 to 100,000

Roche

COBASAmpliPrep/COBASTaqMan 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

SiemansVersant HIV-1 RNA 3.0

(bDNA) Pol Group M: A-H

U.S.: 75-500,000

Ex U.S.: 50-500,000

Abbott RealTime HIV-1 Assay IntGroup M: A-HGroup O, N, P

40 to 10,000,000

bioMerieuxNucliSENS EasyQ HIV-1

v2.0 (RUO US) GagGroup M: A-H 25 to 10,000,000

cps/mL

Biocentric Generic HIV Charge Virale LTRGroup M: A-H

Group N300 to 5,000,000

Cavidi Cavidi ExaVir v.3 RTGroup 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

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

y = 0.9817x + 0.1187R2 = 0.9157

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7

Reference Assay VL(log10)

Liat

Ass

ay V

L (lo

g10)

92% correlation with Abbott m2000 with 75 clinical specimens (clades

A, B, C, and D) Training took 10 minutesEasy to useAssay takes 60 minutes

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 cartridgeThe cartridge is processed by a compact, battery driven instrument

0

1

2

3

4

5

0 1 2 3 4 5 6

log cp/ml COBAS pE

log

cp

/10

µl I

MI w

bE

IMI CLONDIAG HIV VL TestData generated on 1 ml of EDTA Plasma (COBAS Ampliprep/Taqman) versus 10 µl of Whole Blood (IMI’s prototype assay)

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)

donors receiving HAARTtherapy naïve donorsblood viral load equals plasma viral load———

IMInegative

IMIpositive

COBAS plasma negative

73 (28 %) 56 (22 %)

COBAS plasma <40 cp/ml

8 (3 %) 21( 8 %)

COBAS plasma positive

6 (2 %) 94 (36%)

SAMBA HIV-1 POC Test

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

SAMBA HIV-1 POC Test

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

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

Comparison of the Simple Amplification-Based Assay (SAMBA) HIV-1 Test with Commercially Available HIV-1 Nucleic Acid Tests

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 forDiagnosis of Acute Pediatric HIV Infection

Plasma volume: 25L

Immune Disruption: 90oC heat shock

Assay Steps:1. Add 25L plasma to 75L 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 TestUnder Development

1. Separate plasma

2. Pretreat sample in processor

Whole blood volume: 80L

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

Assay Procedure

“Cheap, fast, or accurate. Pick 2”

Cheap:

(< $ 5 USD)

Fast:

< 60 min

Accurate:

Sensitivity: > 95%

Specificity: > 98%

IQuum ??????

Inverness ?????? ???????

SAMBA ?????? < 2 hrs

CIGHT p24

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 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

• 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