AACC HIV testing

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New HIV Tests and Algorithm: A change we can believe in

Esther Babady, PhD, D (ABMM)Memorial Sloan-Kettering Cancer Center

New York, New York

Memorial Sloan-Kettering Cancer Center

Learning Objectives

After this presentation you should be able to:List new methods used for diagnosis of HIV

infections

Explain the advantages of new methods in diagnosis of HIV infections

Describe the most recent CDC/APHL laboratory testing recommendations for HIV diagnosis


Case

68 yo woman

Developed a large parotid mass on neck

Radiologic studies: Abnormal

Fine needle aspirate of parotidEnlarged, atypical cells and abundant necrosis

Suggestive of B cell lymphoma

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Case

Non-Hodgkin’s Lymphoma confirmed

Pre-chemotherapy Infectious Diseases EvaluationHepatitis serologies

HIV serologies


Case

Tests Results Reference range

Hepatitis B surface antigen Negative Negative

Hepatitis B surface antigen antibody

Positive Positive*

Hepatitis B core antibody (Total) Positive Negative

Hepatitis B core antibody IgM Negative Negative

Hepatitis B quantitative PCR Negative [20-170,000,000 IU/mL]

Hepatitis C antibody Negative Negative

HIV-1 and HIV-2 antibodies Negative Negative



HIV-1 quantitative PCR 120,000 [20-10,000,000 copies/mL]

Case

Patient admitted 1 week later

Started on chemotherapy protocol


HIV-1 and HIV-2 antibodies

Negative Negative

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http://www.cdc.gov/hiv/default.html


Background: The virus

Enveloped positive-sense ss RNA virus

Retroviridae Lentivirus

~100 nm diameter

9.7 kb genome 5’ and 3’ LTR, 9-10 genes

http://www.niaid.nih.gov/factsheets/graphics/howhiv.jpg


Background: HIV Phylogeny

Major (M)

HIV-1Outlier (O)

HIV-2 B

A

“Pending” (P)

Non M/O (N)

C,D,E,F,G,H

Circulating recombinant forms

Groups

A, B, C, D, E,F, G, H, J, K

Subtypes

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Background: HIV Distribution

Subtypes Geography

Subtype A West Africa

Subtype B North America and Europe

Subtype C South Africa and S/E Asia

Subtype D Eastern and central Africa

Subtype F Central Africa, South America and Eastern Europe

Subtype G Africa and Central Europe

Subtype H Central Africa

Subtype J Africa and Caribbean

Subtype K DRC and Cameroon

HIV-2 West Africa

McCutchan, F.E. JMV 2006


Background: Stages of Infection

Acute

Flu-like symptoms

Highly infectious

Weeks

Established

Mild or No symptoms

Infectious

Years

AIDS

Low CD4 count

Opportunistic infections

Years

Naif, H.M. Infect Dis Rep. 2013


Background: Epidemiology

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Background: Epidemiology


Trends in Annual Age-Adjusted* Rate of Death Due to HIV Infection, United States, 1987−2010

Note: For comparison with data for 1999 and later years, data for 1987−1998 were modified to account for ICD-10 rules instead of ICD-9 rules.

*Standard: age distribution of 2000 US population


Stage 3 (AIDS) Classifications and Deaths of Persons with HIV Infection Ever Classified as Stage 3 (AIDS), among Adults and Adolescents, 1985–2012—United States and 6 Dependent Areas

Note. All displayed data have been statistically adjusted to account for reporting delays, but not for incomplete reporting. Deaths of persons with HIV infection, stage 3 (AIDS) may be due to any cause.

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Need for new recommendations?

1989 2015


Need for new recommendations?

Better tests

Simpler tests

More commercially available tests

More treatment options


CDC and APHL. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, 2014. Accessed [04/2015]

Laboratory testing sequence

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Laboratory testing: Immunoassays

Utility:Screening

Diagnostics

Additional testing: Acute vs Established

5’LTR 3’LTRgag tatnef

pol vif/vpr/tatrev/vpu

env


Laboratory Testing: Immunoassays

HIV-1/2 IgG abViral culture cell lysates

Synthetic/Recombinant ag

HIV-1/2 IgM/IgG ab

HIV-1 IgG ab1st generation

2nd generation

3rd generation

4th generationHIV-1/2 IgM/IgG ab

p24 ag

Viral culture cell lysates




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Laboratory Testing: Western blot

Cambridge Biotech HIV-1 Western Blot Kit PI


Laboratory Testing: Rapid HIV tests

Rapid test: CLIA waivedManufacturer Test Methods Target Date

Alere Scarborough

Alere Determine™ HIV-1/2 Ag/Ab Combo

Lateral flow Immunoassay

HIV-1HIV-2p24

2013

Bio-Rad Laboratories, Inc

Geenius HIV 1/2 Supplemental Assay

Immuno-chromatograph

HIV-1HIV-2

2014

http://www.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/PremarketApprovalsPMAs/UCM149971.pdf

Accessed 4/2015


Laboratory Testing: Rapid HIV tests

Manufacturer Test Methods Target Date

Bio-Rad Laboratories, Inc.

Multispot HIV-1/HIV-2 Rapid Test

Immuno-concentrationMicroparticle

HIV-1HIV-2

2004

OraSure Technologies

OraQuick ADVANCE® Rapid HIV-1/2 Antibody

Lateral flow Immunoassay

HIV-1HIV-2

2002


Accessed 4/2015

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Laboratory Testing: ImmunoassaysManufacturer Test Methods Generation Date

cleared

Abbott Diagnostics

Architect HIV Ag/Ab combo

ChemiluminescenceMicroparticle

Immunoassay

4th 2010

Bio-Rad Laboratories, Inc.

GS HIV Combo Ag/Ab

EnzymeImmunoassay

4th 2011

Ortho Clinical Diagnostics

Vitros Anti-HIV 1+2

ChemiluminescenceImmunoassay

3rd 2008

Siemens Healthcare diagnostics

Advia Centaur HIV 1/0/2

ChemiluminescenceImmunoassay

3rd 2006


Accessed 4/2015


Laboratory Testing: Molecular tests

UtilityDiagnosis of HIV infection (Qualitative)

Baseline viral load before initiation of treatment (Quantitative)

Monitoring of response to antiviral treatment(Quantitative)

5’LTR 3’LTRgag tatnef

pol vif/vpr/tatrev/vpu

env



Examples of Qualitative Molecular Methods

Manufacturer Test Methods LOD

Hologic Aptima HIV-1 RNA qualitative assay

TMAHPA

100 copies/mL


Accessed 4/2015

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Manufacturer Test Methods Linear range

Abbott Molecular Realtime HIV-1 RT-PCR 40-10,000,000 copies/mL

Roche Molecular Cobas Ampliprep/Cobas Taqman quantitative HIV-1 v.2.0

RT-PCR 20-10,000,000 copies/mL

COBAS® TaqMan® HIV Test v2.0 For Use With The High Pure System

RT-PCR 34- 10,000,000 copies/mL

Cobas Amplicor HIV Monitor v1.5

RT-PCR 50- 75,000 copies/ml400- 750,000 copies/ml

Siemens VERSANT HIV 3.0 Assay (bDNA)

bDNA signal amplification

75 -500,000 copies/mL


Accessed 4/2015


Laboratory Testing: Molecular Tests

Highly specific and excellent sensitivity

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8Exp

ecte

d v

iral

load

s lo

g IU

/mL

Measured viral loads log IU/mL

LOD

LLOQ

ULOQ


Laboratory Testing: Studies

Comparison of WB to a rapid antigen test to on specimens reactive by a 4th generation IA

Philadelphia, PA

Accuracy

Rapid antigen test vs WB: 98.2% vs. 57.9%

Specificity

Rapid antigen test vs WB: HIV-2 misidentified as HIV-1 by WB.

Cardenas, A.M. et al., Journal of Clinical Virology 58S1 (2013), e97–e103

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Comparison of a rapid antigen test to WB, 3rd and 4th

generation immunoassays.

Commercial panels (HIV-1 and HIV-2)

Seroconversion Rapid antigen test: 7-15 days earlier than WB.

Accuracy Rapid antigen test was reactive in 99.0% and 92.5% of 3rd

and 4th generation

Masciotra, S. et al., Journal of Clinical Virology 58S (2013) e54– e58



4th generation HIV Ab/Ag, WB and HIV-1 RNA

HIV treatment center: Amsterdam, Netherlands

~5,000/years over 10 years: 75 cases Ab/Ag positive, WB negative, HIV-1 RNA not

detected

7 cases Ab/Ag positive, WB negative, HIV-1 RNA detected

6 cases Ab/Ag negative, WB negative, HIV-1 RNA detected

Sensitivity/specificity: 99.6%/99.8%.

Jurriaans, S. et al., Journal of Clinical Virology 52S (2011), S67-S69



Comparison of 3rd and 4th generation IA

Multi sites study: CA and WA

Seroconversion 4th generation vs 3rd generation: 7-11 days earlier

Specificity 4th generation vs 3rd generation: 99.7-100%

Mitchell, E.O. et al., Journal of Clinical Virology 58S1 (2013), e79-e84

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Comparison of WB to RAT and NAAT.

11 U.S. public health laboratories

570 remnant 3rd gen IA reactive, HIV WB neg or indeterminate.

~90% confirmed HIV negative

~10% HIV positive (~3% acute HIV and 0.2% HIV-2).

Linley, L. et al., Journal of Clinical Virology 58S1 (2013), e108-e112


Laboratory testing: Genotyping

Utility:Monitoring HIV-1 resistance

Baseline before start of antiretroviral therapy (ART)

Guide alternative ART selection in cases of drug therapy failure


Laboratory Testing: Genotyping

Examples of Genotyping assays

Manufacturer Test Methods LOD Target

Abbott Molecular Viro Seq HIV-1 sequencing system

Sequencing 2000 copies /mL ProteaseRT

Siemens Molecular Diagnostics

TRUGENE HIV -1 Genotyping Assay

Sequencing 1000 copies/mL

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U.S. Preventive Services Task Force

http://www.uspreventiveservicestaskforce.org/Page/Topic/recommendation-summary/human-immunodeficiency-virus-hiv-infection-screening


CDC HIV Testing Guidelines

1989:Testing for HIV-1 immunoassays and confirmation

with HIV-1 WB or IFA (MMWR v38, 1989)

1992:Testing for HIV-2 antibodies (MMWR v41, 1992)

2004:Use of HIV-1 WB/IFA to confirm reactive rapid test

results (MMWR v53, 2004)


CDC HIV Screening Guidelines

2014: Recommended algorithm Initial testing for HIV should be done using 4th

generation immunoassay

Reactive screen should be confirmed with an Ab test that differentiates HIV-1 and HIV-2

If reactive samples do not confirm, additional testing with a HIV-1 NAT

Algorithm should be applied to reactive rapid tests


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CDC HIV Screening Guidelines

2014: Alternative algorithmsReactive 3rd generation immunoassay followed by

recommended algorithm

Reactive WB/IFA followed by HIV-1 NAT and HIV-2 ab test if necessary

Reactive 4th generation IA followed by HIV-1 NAT

Nonreactive 3rd and 4th generation IA followed by HIV-1 NAT



New Testing algorithm



Reporting results

CDC guidelines, 2014

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Conclusions

Several, new, sensitive and user-friendly assays are now available for accurate diagnosis of HIV

Recent CDC/APHL recommendations use combinations of these new methods to identify more cases of acute HIV infections


Self-Assessment Questions

1. The new CDC/APHL recommends that a reactive 4th

generation immunoassay be followed by testing with which

of the following tests?A. HIV-1/HIV-2 ab C. Western Blot

B. HIV-1 RNA D. HIV-2 RNA

2. Which of the following HIV antigens is detected by antibody/antigen combo assays?A. p24 C. gp120

B. p55 D. gp160


Self-Assessment Questions

3. Use of the new CDC/APHL HIV testing algorithm improves detection of acute HIV?A. True

B. False

4. Which of the following test is reactive during the eclipse period of HIV infection?A. qualitative HIV-1 RNA C. Both of the above

B. 4th generation IA D. None of the above

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