early infant diagnosis: current tools and prospects of point of care technology
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Early Infant Diagnosis: 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 - PowerPoint PPT PresentationTRANSCRIPT
Early Infant Diagnosis: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
Acknowledgments
• Dr. Shuqi Chen, IQuum• Dr. Robert Coombs, Univ of Washington• Dr. John Gerdes, Micronics• Dr. Jeanne Jordan, George Washington Univ • Dr. David Kelso, Northwestern Univ• Dr. Helen Lee, Univ of Cambridge• Dr. Laura Mazzola, WAVE 80• Dr. Christopher Myatt, mBio Diagnostics• Dr. Avi Pelossof, Inverness• Dr. Michael Pollack, Advanced Liquid Logic
Desirable Qualities of a POC Diagnostic Test
Rapid (< 1 hour) Sensitive (how sensitive? > 95%) Specific (how specific? > 98%) Inexpensive (< $5/test) Simple
Equipment – battery operated, few moving parts, small footprint
Technique – minimal training required Robust - No cold chain requirement Commercially available CE marked/FDA cleared
Desirable Qualities of a POC Diagnostic Test
“Cheap, fast, or accurate. Pick 2”
(Dr. Bill Rodriguez, Harvard Univ, Nov 16, 2009)
HIV DNA and Total Nucleic Acid Assays
• Roche AMPLICOR HIV DNA assay, v 1.5 is the gold standard – Has been successfully introduced and implemented
in many countries around the world– Can use whole blood pellets or Dried Blood Spots
(DBS)
• Roche Qualitative Total Nucleic Acid Assay has been introduced (Stevens, et al, JCM 2008)– Works on whole blood and DBS– 100% sensitive and 99.7% specific
• Abbott also developing a DNA assay • Both Roche and Abbott assays require large,
expensive new equipment – Probably suitable for large centralized labs
POC HIV DNA Assays• CIGHT, Dr D 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; HIV Diagnostics Mtg, 2010)
• 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
• Qualitative Gen-Probe Aptima – Only HIV RNA assay FDA approved for diagnosis
(though approval is for plasma or serum, not DBS)– Works well with DBS– Very sensitive and specific (Kerr, 2009; Stevens, 2009)– Being used by the State of New York for EID
• 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
Commercially Available HIV-1 Viral Load Assays
Manufacturer Assay Name
Roche Amplicor HIV Monitor v1.5 , COBAS
Roche TaqMan HIV-1, version 1.0, 2.0
Siemans Versant HIV-1 RNA 3.0 (bDNA)
Abbott RealTime HIV-1 Assay
bioMerieux NucliSENS EasyQ HIV-1 v2.0 (RUO US)
Biocentric Generic HIV Charge Virale
Cavidi Cavidi ExaVir v.3
POC RNA AssaysUniv of Cambridge & Diagnostics for the Real
World – isothermal amplification with visual detection by dipstick, LoD 75 cp/mL using 250uL plasma, <90 min
IQuum – realtime PCR, LoD – ~100 cp/mL, 1 hr, 200 uL plasma
Inverness – microarray, realtime detection,10 uL whole blood
• Advanced Liquid Logic - based on digital microfluidics
• Wave 80 – assay based on bDNA assay
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• Semi-quantitative VL assay with cut-off of 1,000 cp/mL• Qualitative assay appropriate for EID • 250 uL plasma - limit of detection 75 cp/mL
100 uL whole blood - limit of detection 400 cp/mL• Robust – no cold chain required, can be battery
operated• Simplified sample preparation chemistry• Sample prep and amplification/detection equipment
not linked currently• Little training required• Field testing in resource-limited settings in September
2010 • Clinical trial for regulatory approval in 2011
LIAT™ Quantitative POC HIV Assay• 200 uL plasma sample input (haven’t tested whole
blood yet)• Realtime PCR• 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
•Can be battery operated•Very simple training and operation•Add 200 uL plasma to cartridge, cap, and insert in instrument
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 5 minutesEasy to useAssay takes 60 minutes
Fiscus, unpublished data 2010
IMI’s CLONDIAG HIV Viral Load Point-of-Care Test
Can use 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 Viral Load Test1 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———
N=258
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
Assay Steps:
1. Add 25L plasma to 75L buffer2. Heat in water bath at 90oC for 4 min3. 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 babies < 6 months of age• 53% from infants < 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%
Whole blood
Robust
(battery operated and
no cold chain)
IQuum ????? In develop-
ment
Needs cold chain
Inverness ????? ?????
SAMBA $10-20?
< 90 min
CIGHT p24
Centralized vs POC Testing
Centralized Testing using DBS
• Can be implemented now• Better control on training,
supply logistics, internal and external QA
• 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 VL assay
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 Continuous QA of POC facilities
Confirmation of all positives at a central/reference lab