Monica Parker, Ph.D. Director, Bloodborne Viruses ...€¦ · Director, Bloodborne Viruses Laboratory Wadsworth Center/NYSDOH 2 ... OraQuick Advance Clearview Complete HIV 1/2 ...

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HIV Diagnostics:New Technologies and Strategies

Monica Parker, Ph.D.

Director, Bloodborne Viruses Laboratory

Wadsworth Center/NYSDOH

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Objectives

• Review the principles of HIV diagnostic testing

• Describe technologies that aid in earlier and more accurate detection

• Present new testing strategies being proposed for HIV diagnostics

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HIV

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Virologic Markers of HIV Infection

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Characteristics of HIV Infection

• Acute/Primary Infection– Ab to virus not detectable (window period)

– Viral RNA detectable by about day 10

– Viral Ag (p24) detectable 5-7 days after RNA

• Established Infection– Viral RNA levels decrease and level off

• May be undetectable, if antiretroviral treatment

– Ab detectable at 3 to 6 weeks post infection

– Ag (p24) may become undetectable due to immune complex formation

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1989

• CDC released recommendations for a sequential, two-test algorithm for diagnosis of HIV-1 infection.

– Screen for antibodies to HIV using an enzyme immunoassay (EIA)

– If repeatedly reactive by EIA, conduct a more specific supplemental test

• Western blot (WB)

• Indirect immunofluorescence assay (IFA)

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2010

• The CDC recommendation from 1989 is still the official stance

• Testing methods have moved forward

• More test options exist today

• Alternative strategies are under evaluation

Antibody Tests

The Standard for HIV Diagnostics

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Antibody Screening Tests

• Enzyme Immunoassays (EIA, ELISA)

• Chemiluminescence Immunoassays (CIA)– Over time, sensitivity has increased

– Repeatedly reactive = reactive on at least two screening tests

– Supplemental testing is required to “confirm”infection

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Enzyme Immunoassay (EIA)1st/2nd Generation

• Microwell plates coated with HIV antigens (Ag)

• Ag = Viral lysate, recombinant or peptide

• Serum/plasma -IgG + enzyme substrate color optical density (OD) reading

-IgG

IgG-HIV

HIV Ag

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EIA: 3rd Generation

• Ag-Ab-Ag sandwich technique

• Multiple HIV Ag: HIV-1(envelope & core), HIV-2, group O

• Increased sensitivity, earlier detectionHIV Ab in

blood

IgG IgM

Ag-enzyme

+

HIV Ag on test surface

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Random Access Platform

• Multiple analytes on the same instrument

• Reagents loaded and stored on board

• Controls run at the beginning of the day

• Load individual specimen tubes & get result in about 1 hr.

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CIA (Chemiluminescence)• Microparticles coated with HIV antigens

• Ag-Ab-Ag sandwich

• Conjugated Ag has chemiluminescent reagent

• Activate and emit light (relative light units = RLU)

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4th Generation Assays

• Combine detection of Ab and Ag (p24)– Ag-Ab-Ag sandwich for Ab detection

– mAb-Ag-mAb sandwich for p24 Ag detection

• Detects HIV infection sooner than Ab-only assays, pre-seroconversion

• Not yet approved by FDA in the US– In use elsewhere in the world

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4th Generation Technology

Blood: Ab + Ag

+

Coated microparticles

Incubate then wash

Tagged MAb + AgChemiluminescence

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Rapid Tests

• 6 rapid tests are FDA-approved for HIV

• 4 are waived under CLIA (whole blood from fingerstick or oral fluid)– Can be performed in limited lab setting

– All detect HIV antibodies

– Interpretation is visual, no instrumentation

• Sensitivity and specificity similar to 3rd

generation EIA tests

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Uni-Gold Recombigen

Multispot HIV-1/HIV-2Reveal G3

OraQuick Advance Clearview Complete HIV 1/2

Clearview HIV 1/2 Stat Pak

HIV Rapid Tests

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Uni-Gold Recombigen OraQuick Advance Clearview Complete HIV 1/2

Clearview HIV ½ Stat Pak

WAIVED(Low complexity)

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Advantages/Disadvantages

• Clients get their results right away– When specimen was sent to lab, many clients

never returned for test results

– Post-test return rates: 70-85%

• Some clients get a preliminary positive result that turns out to be false positive

• Preliminary positive result requires supplemental testing to confirm

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Supplemental Tests

• Supplemental tests are designed to be specific (confirmatory)

• Antibody screening tests have progressed and become more sensitive

• Supplemental tests for confirming HIV antibodies have changed little over the years

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Western Blot

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Western Blot Procedure

• Viral proteins separated and blotted

• Strips incubated with serum or plasma

• Antibodies to major HIV proteins bind

• Positive (CDC/APHL): At least 2 of gp120/gp160, gp41 or p24

• Indeterminate: 1 or more bands present, but does not meet criteria for positive

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Interpretation is Subjective

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Indeterminate Western Blot

• Reasons for indeterminate results:– Early infection– Late infection– HIV-2 infection– Other

• Some individuals may exhibit indeterminate results for long periods of time (years) and never seroconvert

• Most indeterminates show only weak reactions to the Gag proteins (mostly p17, p24 and/or p55)

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HIV-2

• Lots of FDA-approved screening assays– Most are combo assays i.e. HIV-1/2

– Need to differentiate when reactive

• One FDA-approved HIV-2 ELISA test

• Multispot rapid test: Differentiates HIV-1 & HIV-2

• No approved confirmatory tests for HIV-2– Research Use Only kits available

• No qualitative or quantitative HIV-2 RNA tests commercially available

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Negative HIV-1 & HIV-2 Positive

Peptide HIV-2

Recombinant HIV-1

Peptide HIV-1

Reactive Control

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Test Sensitivity in Early Infection

Owen et al 2008 JCM 46:1588-95

What AboutNucleic Acid Tests?

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HIV-1 RNA Testing

• Quantitative HIV-1 RNA tests (viral load)– Intended use (FDA): monitoring therapy, prognosis

– Alone, not intended for diagnosis

• Qualitative HIV-1 RNA test– Intended use ( One FDA approved):

– as an aid in the diagnosis of HIV-1 infection, including acute or primary infection

– to confirm HIV-1 infection in an individual whose specimen is repeatedly reactive for HIV-1 antibodies

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Applications for RNA Testing

• Testing symptomatic or recently exposed individuals (AHI)

• Resolving discordant Ab test results:– EIA or rapid test reactive, WB neg or ind

• Screening pooled seronegative specimens (OFF-LABEL USE)

• Early infant diagnosis

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Acute HIV Infection (AHI):Screening pooled plasma by NAT

• Technology used routinely for blood donor screening

• No FDA approved kits for diagnostic use of RNA testing on specimen pools

• Pooled RNA testing after antibody screening has been shown to be feasible in public health setting

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Pool

Testfor

RNA

Deconstruct & Test Individual Specimens

Screening for Acute/Early Infection

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CDC AHI StudyNegative Antibody Screen

Blood specimen to lab

16-specimen poolsQualitative RNA test

RNA negative(HIV negative)

RNA positive pool

Test individual specimensIdentify HIV-positive

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Case Detection Increasedby RNA Testing

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Variable Yield with RNA Screening

• Parallel study was conducted with STD clinic in an upstate NY county

• 5722 HIV antibody-negative specimens screened by pooled RNA testing

• 1 EIA reactive/ WB indeterminate/RNA positive result

• No true AHIs detected

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Recap on Testing Capabilities

• More FDA-approved tests available– Including several rapid tests

• Screening tests have increased sensitivity; Western blot has not

• Rapid tests get neg and prelim pos results to clients immediately; still need to confirm

• RNA tests may be used in the detection of acute/early infection

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Updating the HIV Diagnostic Testing Strategies

• 2006: Two workgroups convened to develop proposals for the best combination of assays to determine HIV infection status in laboratory and point-of-contact (POC) settings

• Dec 2007:HIV Diagnostics Conference:– 5 laboratory testing strategies proposed

– 4 POC strategies proposed

Laboratory-based Testing Strategies

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Status Report

• April 2009: HIV Testing Strategies, A Status Report released by CDC and APHL– http://www.aphl.org Infectious Diseases

• What are the false positive and false negative rates of these strategies?

• Are we ready to do away with the Western blot?

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Moving Forward

• March 2010: HIV Diagnostics Conference– Progress was made towards developing a unified

laboratory algorithm– Consideration was given to early detection, accuracy

and cost– Proposed strategy:

• two different antibody screening tests• NAAT for discordant cases• No role for WB

• Implications for HIV case reporting and surveillance?

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Point-of-Contact Strategies

• 2002: 1st HIV rapid test FDA approved for use at the POC

• CDC: Must confirm with WB or IFA– Intended use statements: “Suitable for use in multi-

test algorithms designed for statistical validation of rapid HIV tests”

• Multi-rapid test strategies are being evaluated

• Additional rapid test used to “verify” results of initial test, not “confirm” infection

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Each Test is Different

•25 / box• 20 / box• 25 or 100 / box

• Finger stick only• Finger stick only• Oral or Finger stick sample

• Must be read by 20 minutes

• Must be read by 12 minutes

• Must be read by 40 minutes

•Test storage temp (46-86°F)

• Test storage temp (35-80°F)

• Test storage temp (35-80°F)

• 15 minutes• 10 minutes• 20 minutes

• HIV1 & HIV2• HIV1 Only• HIV1 & HIV2

ClearviewUni-GoldOraQuick

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More Differences

• Clinic temperature to test (64 to 86°F)

• Clinic temperature to test (59 to 80.6°F)

• Clinic temperature to test (59 to 99°F)

• Controls good for 2 yrs (check expiration date)

• Controls good for 30 days after opening

• Controls good for 8 wks after opening

•Use test stand• Lay flat on table• Use blue stand

• If result invalid, retest with 1 device

• If result invalid, retest with 2 devices

• If result invalid, retest with 1 device

• Insert device into buffer vial

• Wash solution added to device

• Device added to developer solution

• Sampler tip collection• Pipette collection• Loop collection

ClearviewUni-GoldOraQuick

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Questions

• Can multiple rapid tests be reliably performed in sequence at the POC?– Most data have been collected retrospectively

in a controlled lab setting

• What is the best sequence of rapid tests for multi-test algorithms?

• Collection of real-life data from rapid testing programs is underway

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More Questions

• Rapid test → Preliminary positive → Refer to care– Many will go, some will not

• Lab confirmation = Lab reporting

• Will we see a decrease in case reports?

• Will active surveillance be needed

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Summary

• The number and variety of HIV diagnostic tests have expanded greatly over time

• The capacity for early detection has improved

• New strategies are needed to fully take advantage of the testing options

• National recommendations are anticipated soon