Diagnosis of HIV Infection

Theodoros Kelesidis, MD, PhD

Assistant Professor of Medicine Attending Physician Ronald Reagan Medical Center

Division of Infectious DiseasesDavid Geffen School of Medicine

February 12, 2014

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HIV in the U.S.

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.

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

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Why provide HIV testing?

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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Human Immunodeficiency Virus

RNA virus

Surface envelope proteins

Matrix proteins Capsule proteins RNA

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Evolution of HIV infection

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HIV tests

Detect human antibodies to surface, matrix and capsule proteins

Detect HIV antigen p24 (a nucleocapsid protein) and gp 120 and gp 41 (envelope proteins).

Detect viral nucleic acid (NAT) a) RNA b) DNA integrated within cells

Viral culture in patient PBMC

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HIV antibody tests

Enzyme immunoassays (EIA) 1st generation—IgG anti-HIV antibodies 2nd generation—synthetic antigen + HIV-2 3rd generation—IgM and IgG anti-HIV antibodies 4th generation—p24 HIV antigen + IgM and IgG anti-HIV

antibodies

Western blot Immunofluorescent assay

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What is an ELISA test?

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).

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What is an ELISA test?

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

Scientist developed an anti-HIV antibody antibody. So this new antibody sticks to the 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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HIV 1st Generation Antibody test

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Problems with HIV 1st Generation Antibody test

• It only detects antibodies to the HIV 1 virus, not HIV 2.• It only detects the IgG antibodies which can take some

time to be produced in the body up to levels which are detectable testing window period.

• The antigen purification from viral lysates was not very good high level of false positives need for confirmation by Western Blot

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HIV 2nd Generation Antibody test

• Includes HIV-2 antigen

• Uses synthetic antigens better antigen production techniques that improved the false positive rate

• Same problems with 1rst generation tests

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HIV 2nd Generation Antibody test

Rapid test

• OraQuick Rapid HIV-1/2 Antibody Test

• Clearview HIV 1/2 Stat Pak • Reveal G3 Rapid HIV-1

Antibody Test • Uni-Gold Recombigen HIV Test • Multispot HIV-1/HIV-2 Rapid

Test • Clearview Complete HIV 1/2

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HIV 3rd Generation Antibody test

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

Detect all kinds of antibody of HIV (including IgM)

Improved sensitivity and specificity as well.

The main laboratory-based diagnostic ELISA test worldwide now.

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HIV 4th Generation Antibody test

The method of 4th generation of HIV ELISA test is that HIV antigen and p24 antigen coat the vector simultaneously

Detect the HIV P24 and HIV antibody in the sample at the same time.

So, it could be used for detection for the samples of window period which shows positive in HIV P24 antigen tests but is not transformed into HIV infection.

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HIV Western blot

• 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, 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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Reasons for false negative results

• Acute infection• Failure to detect certain HIV subtypes EIAs in USA and Europe detect all M subtypes but do not consistently detect other groups (O, P)4th generation can detect O subtypes more reliablyThe newly detected P subgroup can be detected only with nucleotide sequencing • Immune dysfunction due to immunosuppression (e.g

medications, malignancy)• Delay in seroconversion following early initiation of

antiretroviral therapy • Fulminant HIV infection e.g presentation with OIs like PCP

 

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Reasons for indeterminate results

• Indeterminate tests usually result from a positive EIA and a single band on Western blot (usually p24).

• Causes of indeterminate results include A) partial seroconversion during acute HIV infection B) advanced HIV infection with decreased titers of p24 antibodiesC) infection with HIV-2.D) Cross-reacting alloantibodies from pregnancyE) Autoantibodies (collagen-vascular diseases, autoimmune diseases,

and malignancy)F) Receipt of an experimental HIV-1 vaccineG) Influenza vaccination

 

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In an IFA test, HIV antigen is mixed with a fluorescent compound and then with a sample of the patient's blood. If HIV antibody is present, the mixture will fluoresce when examined under ultraviolet light.

HIV Immunofluorescent assay

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HIV-RNA Qualitative Test

Used to diagnose HIV infection Resolve indeterminate HIV-antibody results Diagnosis of neonatal HIV infection, in patients with

indeterminate serologic tests, or in those who may be in the "window period" of HIV seroconversion

HIV PCR RNA – virus. DNA – pro-virus. There is more data for using the RNA as a diagnostic test. Also, RNA is detectable earlier than DNA

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HIV-RNA Quantitation

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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HIV-1 Proviral DNA Detection

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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HIV-1 Culture

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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HIV-1 Genotypic Resistance Assay

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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Case 1

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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Case 2

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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Case 3

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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Case 4

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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Case 5

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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Case 6

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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Case 7

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

Thank You

tkelesidis@mednet.ucla.edu

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