EID - Current tools and prospects of point of care technology Susan A. Fiscus, Ph.D. University of North Carolina at Chapel Hill
Mar 31, 2015
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