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การประชุมวิชาการด ้านการตรวจทางห ้องปฏิบต
ั ก
ิ ารเอชไอวี/เอดส ์
ระดับชาติ ครัง้ ที่ 3
วันที่ 11-13 กรกฎาคม 2554
ณ โรงแรมฮอลิเดย์อน
ิ น์ รีเจนท์ บีช ชะอา จ.เพชรบุรี
New Technologies and Innovations in
Laboratory Support of HIV/AIDS Care
and Treatment
Sakchai Dettrairat
HIV Laboratory
Division of Clinical Immunology
Department of Medical Technology
Faculty of Associated Medical Sciences, CMU
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
HIV RNA
HIV p24 Ag
HIV Antibody
Day 0
Day 11
Day 16
Day 22
5 Days
6 Days
11 Days
http://depts.washingto
n.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
Sandwich ELISA for detection of HIV Ab
Substrate
Enzyme conjugated
HIV Ag
E
Color Product
E
E
IgG Ab
E
E
E
IgM Ab
rHIV Ag and
synthetic peptides
Env/Core Ags
Fourth generation Sandwich ELISA
for detection of HIV Abs and Ag
Substrate
Enzyme conjugated
HIV Ag
E
E
E
E
Anti-HIV
Ab
rHIV Ag/
peptide
Color Product
Enzyme-conjugated
Anti-p24 Ab
E
E
p24 Ag
Anti-p24 Ab
Fourth-generation combination
EIAs
HIV Rapid Tests
• Qualitative test to detect
HIV-1/2 Abs
• Simple/Moderate Complexity
• Small sample volumes
• Serum/Plasma/Whole blood
• Visual Reading
• Results in 10-15 minutes
Lateral flow strip
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
Control line
(Anti-HIV)
Test line
(HIV Ag)
Colloidal
Goldconjugated
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
HIV testing Algorithms in
Adults and Children >18 M
A1
A1- (NR)
A1+ (R)
A2
A1+, A2-
A1+, A2+
Repeat A1 and A2
A1-, A2-
A1+, A2-
A1+, A2+
A3
A1+, A2+, A3Anti-HIV negative
Indeterminate
Retest at 2 W/3 and 6 m
A1+, A2+, A3+
Anti-HIV Positive
Retest sample 2
HIV testing Algorithms in Children <18 M
Age <12 m
Age 12-18 m
HIV viral testing at >1 m
Ab testing
Positive
Negative
Repeat testing
within 2-4 w
Repeat testing
at age 4 m
Positive
Negative
Positive
HIV infection
Nonreactive
Ab testing at 18 m
Negative
Repeating testing
at age 4-6 m
Positive at 2nd
and 3rd testing
Reactive
Reactive
Nonreactive
Discordant at 2nd
and 3rd testing
Anti-HIV
positive
Anti-HIV
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
Coming Soon
Control bar (anti-HIV)
p24 bar (avidin)
Ab bar
(rHIV Ag + synt peptides)
Conjugate pad
(Selenium colloid-HIV Ag/
Biotinylated anti-p24+
Selenium colloid anti-p24)
B
4th Generation Immunochromatograpic test
for detection of HIV Ag/Ab
Coming Soon
Control bar (anti-HIV)
p24 bar (avidin)
B
Ab bar
(rHIV Ag + synt peptides)
B
Conjugate pad
(Selenium colloid-HIV Ag/
Biotinylated anti-p24+
Selenium colloid anti-p24)
B
Patient sample
Determine HIV-1/2 Ag/Ab Combo
Coming Soon
Control bar
p24 Antigen bar
Antibody bar
Selenium
colloid
Conjugate pad
The 4th generation of one step HIV
antigen and antibodies to HIV-1/2 Test
Coming Soon
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 Schematic
http://www.theengineer.co.uk/sectors/medical-and-healthcare/news/lab-onchip-device-promises-hiv-diagnosis-in-10-minutes/1008004.article
New HIV Testing Algorithms ?
http://www.aphl.org/aphlpro
grams/infectious/hiv/Docum
ents/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/market
dynamics/publications/unitaid_md_te
chnical_report_diagnostics_landscape
_web.pdf
CD4 T Cell Counts
CD4 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 Counts
Factors 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/201007/uoc--nmh071610.php
Zyomyx CD4 Test
Principle:
• CD4 reagents bind to CD4 Tcells
• selectively transfer them
into a secondary liquid
medium
• concentration of the reagent
binded CD4 T-cells in a
volumetric compartment
• determination of the CD4 Tcell 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 1520 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
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