Jeffrey D. Klausner, MD, MPH

Professor of Medicine and Public HealthAttending Physician Ronald Reagan Medical Center

Center for World Health and Division of Infectious DiseasesDavid Geffen School of Medicine

Department of EpidemiologyKarin and Jonathan Fielding School of Public Health

AAHIVU August 2014

Dr. Klausner is a faculty member of the University of California Los Angeles Dr. Klausner is a guest researcher with the US CDC Mycotics Diseases Branch Dr. Klausner is a member of the WHO STD Guidelines group Dr. Klausner is a board member of YTH, Inc, non-profit Dr. Klausner is medical advisor for Healthvana.com

In the past 12 months, Dr. Klausner has received: Travel support for meeting coordination and speaking from Standard Diagnostics, Inc. Research funding or donated supplies from the NIH, CDC, Hologic, Inc., Alere, Inc.,

Chembio, Inc. Cepheid, Standard Diagnostics, Inc., and MedMira, Inc.

JDKlausner@mednet.ucla.edu

An estimated 1,148,200 persons in the United States live with HIV About 50,000 new HIV infections occur in the United States each

year Each year, approximately 16-22 million persons in the United

States are tested for HIV An estimated 38%-44% of all adults had been tested for HIV Approximately 1 in 5 (18%, or 207,600 persons) do not know they

are HIV-infected.

3US CDC, 2012 http://www.cdc.gov/hiv/resources/factsheets/us.htm

Earlier detection leads to improved treatment outcomes HIV-infection status awareness is associated with reduced

transmission risk behavior HIV-infected persons on treatment are less infectious

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SCREENING TEST

High Sensitivity

CONFIRMATION TEST

High Specificity

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RNA virus

Surface envelope proteins

Matrix proteins Capsule proteins RNA

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Detect human antibodies to surface proteins Detect human antibodies to surface, matrix and

capsule proteins

Detect HIV antigen (P24 capsule protein)

Detect RNA Detect DNA, integrated within cells

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1st generation—viral lysate 2nd generation—synthetic antigen + HIV-2 3rd generation—IgM and IgG + HIV-2 4th generation—p24 + IgM and IgG + HIV-2

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Enzyme-linked immunosorbent assay (ELISA or EIA) 1st generation—viral lysate 2nd generation—synthetic antigen and HIV-2 3rd generation—IgM and IgG and HIV-2 4th generation—p24 + IgM and IgG and HIV-2

Western blot Immunofluorescent assay

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ELISA = Enzyme-Linked Immuno Sorbent Assay. This technique is based on the lock and key theory of antibodies.

Antibodies and antigens work like locks and keys. One antibody fits one antigen. Having the antibody means the antigen is also present.

ELISA technique involves placing HIV antigens (locks) on the bottom of a microwell cup

The microwell is then filled with the serum to be tested. If the appropriate anti-HIV antibodies are present (keys), they will stick to

the antigens (locks). High throughput, automated, sensitive

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Since antibodies are proteins too, they themselves are also antigens.

Use anti-HIV antibody antibody to capture back of the first antibody.

This second antibody has an enzyme is attached to it.

When a reactive substrate is added to the mix, the enzyme will turn the substrate a different color (usually red).

If the serum to be tested contains anti-HIV antibodies, the liquid in the microwell will turn red.

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Detects anti-HIV IgG

Includes HIV-2 antigen

Uses synthetic antigens

Window period 6+ weeks

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• OraQuick Rapid HIV-1/2 Antibody Test • Reveal G3 Rapid HIV-1 Antibody Test • Uni-Gold Recombigen HIV Test • Multispot HIV-1/HIV-2 Rapid Test • Clearview HIV 1/2 Stat Pak • Clearview Complete HIV 1/2

The method of 3rd generation of HIV ELISA test is double antigen sandwich

Detects IgM and IgG antibody to HIV

Antibodies to HIV-2, HIV-1 group O Improved sensitivity and specificity The main laboratory-based

diagnostic ELISA test worldwide now

Window period 3 weeks

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Detects antibodies to HIV -1, HIV-2 and HIV-2 O type AND p24 antigen

Used for detection of infection within window period which shows positive in HIV P24 antigen tests but negative for antibodies to HIV

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Western blot Immunofluorescent assayQualitative RNA tests

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• Identifies antibodies against eight HIV-1 encoded proteins: p18, p24, p31, gp41, p51, p55, p65/66, gp120/p160.

• Criteria require antibodies against any two of the following HIV-1 proteins: p24, gp41, or gp120/160.

• Specimens showing reactivity to HIV-1 protein(s), but not fulfilling the criteria for a positive result, are reported as Indeterminate.

• All indeterminate Western blots are further tested in supplemental HIV-1 and HIV-2 specific assays.

• A negative Western blot has no detectable bands, i.e. no antibodies reacting to either HIV-1 or non-HIV-1 proteins.

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Mandel, Principles & Practice of Infectious Diseases, 6th Edition

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HIV Immunoflourescent assay

Used to diagnose HIV infection Resolve indeterminate HIV-antibody results

Manual Lower limit of detection > 30 RNA copies/ ml

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Used to monitor antiviral therapy and to predict disease progression in HIV infected persons.

In conjunction with a positive DNA PCR or a reactive EIA, the RNA quantitation may be diagnostic. High levels of RNA are found during acute infection and in patients who

are more likely to have disease progression. Inhibition of cell-free HIV, as reflected by RNA copy number, is

associated with better CD4 response and clinical response in some patient populations.

The dynamic range for HIV RNA detection by Real-Time PCR is 30 to 1,000,000 copies/mL of plasma.

Often use in newborns and infants for early diagnosis

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The detection of cell associated Human Immunodeficiency Proviral DNA by polymerase chain reaction (PCR) amplification is one of the most sensitive non-serologic methods for confirming HIV infection.

This assay is recommended for confirming HIV infection in the neonate.

HIV DNA PCR may also be used as a supplemental test to determine the significance of an indeterminate HIV Western Blot serology result.

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Culture is an extremely sensitive virologic method for documenting HIV infection, especially in neonates whose serologies are complicated by the presence of maternal antibody

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The assay involves sequencing of the HIV pol gene, after which mutations in the gene can be compared to sequences known to confer resistance to different classes of antiretroviral drugs.

The assay is most useful in patients who lose viral suppression on antiretroviral therapy and should be performed before switches in therapy are entertained.

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Enzyme Immunoassay

(EIA)(run singly)

End; patient isconsidered

negative

non-reactive reactive

Repeat EIA in

Duplicate

Patient is considered

negative

2 non-reactiveresults

1 or 2reactiveresults

Must perform one of the following confirmation tests

IFAimmunofluorescence

assay

WesternBlot

TMA transcription-

mediatedamplification

Positive: patient is HIV+

Negative: patient is HIV-negative

Indeterminate

Result = inconclusive

Re-draw in 2-4 weeks

Most sensitive EIA available

4th GenerationStrongly

recommended(run singly)

HIVNegative

PositiveNegative RepeatIn

Duplicate

2 neg

HIVNegative

1 or2 pos

An HIV-1 / HIV-2 Differentiating TestPos for

HIV-1 Ab

HIV-1Positive

Pos forHIV-2 Ab

HIV-2Positive

Neg

HIV RNA Test No RNA

HIVNegative

(follow-up for HIV-2)

RNA Detected

HIV-1Acute Infection

30 year old bisexual man comes into clinic He has had 15 lifetime partners, never been HIV-tested What test is appropriate?

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22 year old man who has sex with men, methamphetamine user

Last tested HIV-negative 6 months ago History of syphilis What test is appropriate?

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46 year old man diagnosed with HIV-infected 6 years ago Has been on treatment for 3 years but has not had a check

up in a year What test is appropriate?

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17 year old girl had a rapid HIV test that was positive She comes to clinic for testing What test is appropriate?

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17 year old girl had a rapid HIV test that was positive The ELISA test was indeterminate What test is appropriate?

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47 year old man has been on treatment for years but ran out of meds 1 year ago.

About 6 months ago he restarted 2 medications he obtained from his partner.

He has been losing weight and complains of fatigue and fevers

What test is appropriate?

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6 week baby had an HIV-infected mother The mother receive treatment during pregnancy What test is appropriate for the baby?

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JDKlausner@mednet.ucla.edu

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