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

การประชุ�มวิชุาการด้�านการตรวิจทางห้�องปฏิบั�ตการเอชุไอวิ�/เอด้ส์� ระด้�บัชุาต คร��งท�� 3

วิ�นท�� - 1113 กรกฎาคม 2554ณ โรงแรมฮอลิเด้ย์�อนน� ร�เจนท� บั�ชุ ชุะอ(า จ.เพชุรบั�ร�

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