Transcript HIV Testing - UCLA Health

Jeffrey D. Klausner, MD, MPH
Professor of Medicine and Public Health
Attending Physician Ronald Reagan Medical Center
Center for World Health and Division of Infectious Diseases
David Geffen School of Medicine
Department of Epidemiology
Karin and Jonathan Fielding School of Public Health
AAHIVU
August 2014
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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.
[email protected]
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 HIVinfected.
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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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SCREENING TEST
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High Sensitivity
CONFIRMATION TEST
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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
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Detect HIV antigen (P24 capsule protein)
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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
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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.
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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 antiHIV 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
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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 assay
Qualitative 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
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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.
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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.
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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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End; patient is
considered
negative
non-reactive
Enzyme
Immunoassay
(EIA)
(run singly)
Repeat
EIA in
Duplicate
reactive
2 nonreactive
results
1 or 2
reactive
results
Must perform one of the following
confirmation tests
IFA
immunofluorescence
assay
Western
Blot
Patient is
considered
negative
TMA
transcriptionmediated
amplification
Indeterminate
Positive: patient is HIV+
Negative: patient is HIV-negative
Result = inconclusive
Re-draw in 2-4 weeks
Most sensitive
EIA available
Negative
HIV
Negative
Positive
4th Generation
Strongly
recommended
(run singly)
Repeat
In
Duplicate
2 neg
1 or
2 pos
Pos for
HIV-1 Ab
HIV-1
Positive
HIV
Negative
An HIV-1 / HIV-2 Differentiating Test
Pos for
HIV-2 Ab
Neg
HIV RNA Test
HIV-2
Positive
No RNA
RNA
Detected
HIV-1
Acute Infection
HIV
Negative
(follow-up for HIV-2)
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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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[email protected]
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