Theodoros Kelesidis, MD, PhD
Assistant Professor of Medicine
Attending Physician Ronald Reagan Medical Center
Division of Infectious Diseases
David Geffen School of Medicine
February 12, 2014
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.
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US CDC, 2012 http://www.cdc.gov/hiv/resources/factsheets/us.htm
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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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RNA virus
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Surface envelope
proteins
Matrix proteins
Capsule proteins
RNA
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Detect human antibodies to surface, matrix and capsule
proteins
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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
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Viral culture in patient PBMC
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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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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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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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• 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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• 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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• 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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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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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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• 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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• 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 reliably
The 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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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 antibodies
C) infection with HIV-2.
D) Cross-reacting alloantibodies from pregnancy
E) Autoantibodies (collagen-vascular diseases, autoimmune diseases,
and malignancy)
F) Receipt of an experimental HIV-1 vaccine
G) Influenza vaccination
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HIV Immunofluorescent assay
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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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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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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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tkelesidis@mednet.ucla.edu
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