New Technologies and Innovations in Laboratory Support of HIV/AIDS Care and Treatment Sakchai...
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Transcript of New Technologies and Innovations in Laboratory Support of HIV/AIDS Care and Treatment Sakchai...
New Technologies and Innovations in Laboratory Support of HIV/AIDS
Care and Treatment
Sakchai DettrairatHIV Laboratory
Division of Clinical ImmunologyDepartment of Medical Technology
Faculty of Associated Medical Sciences, CMU
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HIV/AIDS Diagnostic Landscape
• Diagnostics for HIV/AIDS can be divided into 3 test categories: – Tests to facilitate initial diagnosis– Tests to stage the patient – Tests to monitor the patient, both before
and after initiation of ART
•
HIV Diagnostic Testing
• - Anti HIV Antibody Test• HIV p24 Antigen Test• Nucleic Acid Amplification Tests (NAT)• Viral Culture
HIV Markers During Early Infection
Theoritical Identify Day 0HIV RNA Day 11HIV p24 Ag Day 16HIV Antibody Day 22
5 Days6 Days 11 Days
http://depts.washington.edu/labweb/Divisions/Viro/HIVTest.htm
- Anti HIV Antibody Tests
Screening Tests EIA or CIA Simple/ Rapid Tests
Confirmatory or Supplemental Tests Western Blot (WB) Immunofluorescence Assay (IFA)
Time during acute infection at which positive results of diagnostic tests can be obtained.
Third generation Double Antigen Sa ndwich ELISA for detection of HIV
Ab
rHIV Ag and synthetic peptides
Enzyme conjugated
HIV Ag
IgG AbIgM Ab
Substrate Color Product
Env/Core Ags
E E EE
EE
Fourth generation Sandwich ELISAfor detection of HIV Abs and Ag
Anti-p24 AbrHIV Ag/ peptide
Enzyme-conjugatedAnti-p24 Ab
p24 Ag
Enzyme conjugated HIV Ag
Anti-HIV Ab
Substrate Color Product
EE
E EE
E
Fourth-generation combination EIAs
Lateral flow stripLateral flow strip
HIV Rapid Tests
• Qualitative test to detect HIV-1/2 Abs
• Simple/ Moderate Comple xity
• Small sample volumes • Serum/Plasma/Whole bloo
d• Visual Reading• - Results in 10 15 minutes
Rapid testing is the method of choice when immediate information is necessary to determine the need for prophylaxis, such as in the labor/delivery, newborn, or post-exposure settings, or when the person who is being tested is unlikely to return for a follow-up visit.
HIV Lateral Flow Strips
HIV Rapid Test or Lateral Flow Strip
for detection of antibodies to HIV-1/2
Test line ( HIV Ag)
Control line -(Anti
HIV)
Colloidal Gold-
conjugated HIV Ag
Patient sample
Thailand HIV testing algorithm for individual diagnosis
• National Guidelines on HIV/AIDS Diagnosis and Treatment: Thailand 2010
• National Guide for HIV Testing. MOPH 2007
Anti-HIV negative
HIV testing Algorithms in Adults and
Children >18 MA1- (NR) A1+ (R)
A2
Repeat A1 and A2
A1+, A2+
A1+, A2+, A3+
Anti-HIV PositiveRetest sample 2
Indeterminate
A1+, A2+, A3-
Retest at 2 W/3 and 6 m
A1
A3
A1-, A2-
A1+, A2- A1+, A2+
A1+, A2-
HIV testing Algorithms in Children <18 M
Anti-HIV negative
Anti-HIV positive
Reactive
Nonreactive
Age 12-18 m
Ab testing
Nonreactive
Ab testing at 18 m
Reactive
HIV infection
Discordant at 2nd and 3rd
testing
Age <12 m
HIV viral testing at >1 m
Negative
Positive
Repeat testing at age
4 m
Repeat testing within
2-4 wPositiv
eNegativ
eRepeating
testing at age 4-6 m
Positive at 2nd and 3rd testing
Positive
Negative
New Technologies and Innovations in HIV Diagnosis
• DPP®HIV 1/2 SCREEN Assay– A Rapid Qualitative Screening Assay for the Detection
of Antibodies to HIV 1/2 in Oral Fluid, Whole Blood (Venous or Finger-stick), Serum & Plasma
– Using colloidal gold protein A conjugate
DPP HIV 1/2 Test Procedure
Interpretation of Test Results
4th Generation Immunochromatograpic test for detection of HIV Ag/Ab
p24 bar (avidin)
Control bar (a -nti HIV)
Conjugate pad(Selenium colloid-HIV
Ag/Biotinylated anti-p24+Selenium colloid anti-
p24)
Ab bar (rHIV Ag + synt peptides)
B
Coming Coming SoonSoon
4th Generation Immunochromatograpic test
for detection of HIV Ag/Ab
B
Patient sample
B
B
Control bar (a -nti HIV)
p24 bar (avidin)
Ab bar (rHIV Ag + synt peptides)
Conjugate pad(Selenium colloid-HIV
Ag/Biotinylated anti-p24+Selenium colloid anti-
p24)
Coming Coming SoonSoon
Determine HIV-1/2 Ag/Ab Combo
p24 Antigen barAntibody bar
Control bar
Selenium colloid
Conjugate pad
Coming Coming SoonSoon
The 4th generation of one step HIV antigen and antibodies to HIV-1/2
Test
Coming Coming SoonSoon
Lab on a chip device promises HIV diagnosis in 10 minutes
• A new diagnostic device promises confirmation of HIV, TB and other conditions in around 10 minutes.
• The ‘lab-on-chip’ device is entirely self-powered, using pressure differences and gravity to separate components of a small blood sample.
SIMBAS Chip Schematichttp://www.theengineer.co.uk/sectors/medical-and-healthcare/news/lab-on-chip-device-promises-hiv-diagnosis-in-10-minutes/1008004.article
New HIV Testing Algorithms ?http://www.aphl.org/aphlprograms/infectious/hiv/Documents/StatusReportFINAL.pdf
APHL & CDC 2009• POC Algorithm 1-4• Laboratory Algorithm
1-5
Laboratory Algorithm 2
Laboratory Algorithm 4
Tests to Stage and Monitor of Patients
• CD4 T Cell Counts• Viral Load Assays • Drug Resistance Tests
http://www.unitaid.eu/images/marketdynamics/publications/unitaid_md_technical_report_diagnostics_landscape_web.pdf
CD4 T Cell CountsCD4 T Cell Counts• used to assess immune status • susceptibility to OIs• need for ART and OI prophylaxis• for defining AIDS (CD4 <200/mm3)Facts about CD4 • Normal adult CD4 count reference ranges vary by lab;
however, ranges are generally within 500 -1500/mm3. • Large individual variability in measurement reflecting
method of determining CD4 count by calculation from 3 measured variables: WBC, % lymphocytes, and %CD.
http://www.hopkins-aids.edu/management/laboratory_testing/ cd4_cell_count.html?contentInstanceId=8279
CD4 T Cell Counts
Facts about CD4 • CD4% less variable than absolute CD4 count;
– within-subject CV is 18% for CD4% vs 25% for Abs CD4 count.
• Approximate corresponding values for CD4 count and CD4% are: – >500 ~ >29%, 200-500 ~ 14-28%, <200 ~ <14%.
• Specimens must be processed within 18 hrs of collection for optimal accuracy
CD4 T Cell CountsFactors that Affect CD4 Count • Seasonal and diurnal variation (lowest at 12:30 PM,
highest at 8:30 PM), surgery, viral infections, tuberculosis. • Decreases with some medications (e.g. corticosteroids,
especially with acute use, less pronounced with chronic use; PEG-IFN, IFN; cancer chemotherapy).
• Acute changes in CD4 more often due to redistribution of CD4 in lymphatics, spleen and bone marrow.
• Splenectomy causes abrupt, prolonged increase in CD4. In asplenic pts, CD4% provides more accurate assessment of immune status
• Sex, race, psychologic stress, & physical stress have minimal effect on CD4. Pregnancy leads to hemodilution & a small decline in CD4 count, but no decline in CD4%.
CD4+ T Cell Counting
• CD4 Technologies–Flow Cytometry (DPT & SPT)–BD FACSCount (SPT)
–Point-of-Care CD4 Technologies
Point of Care CD4 Technologies
• PointCARE• LabNow• Pima Analyzer• Daktari CD4 System• Zyomyx CD4 Test• CD4 Rapid Test
LabNow CD4
Pima Point of Care CD4• Absolute CD4 detection• Fingerstick or Venous whole blood• Low sample volume – 25μL• Closed cartridge system• No cold-chain requirements• Fluorescent detection of CD4+
cells• 20min/day internal QC procedure• Sample analyses 20 minutes
Daktari CD4 HIV Diagnostic Instrument
• Microfluidic cell• Electrochemical
sensing (lysate impedance measurement)
Lab on a chip device for HIV testing
• Novel microfluidic HIV test is quick and cheap
• Analysis of CD4 and CD8 T-cell numbers, the CD4/CD8 ratio and three secreted cytokines took only seconds.
http://www.eurekalert.org/pub_releases/2010-07/uoc--nmh071610.phphttp://www.eurekalert.org/pub_releases/2010-07/uoc--nmh071610.php
Zyomyx CD4 TestPrinciple:• CD4 reagents bind to CD4
T-cells • selectively transfer them
into a secondary liquid medium
• concentration of the reagent binded CD4 T-cells in a volumetric compartment
• determination of the CD4 T-cell volume by reading the length of a dark visible line
• measurement of length as a function of CD4 cell.
Zyomyx CD4 Test
Zyomyx CD4 Test
2012
STATUS Quick Screen CD4 EQ 200
• Rapid, cost-effective CD4 test
• Cutoff of 250+/- 25 CD4 cell equivalents
• Uses only 3drops of plasma or serum
• Results available in 15-20 min
OnSight™ – CD4 STATUS Rapid Test
• Positive - test line visible which indicates CD4 EQ’s of greater than 200 (+/- 25), or
• Negative - no test line visible which indicates CD4 EQ’s of less than 175 (+/- 25)
Retro-Status HIV/CD4 350 Rapid Test
• HIV/CD4 Dual test• Detects HIV1/2 Abs• Cutoff of 350 CD4
cells/millitre • Uses few drop of sample• Results available in 20
minutes
Thank you for your attention