1. Diagnosis of HIV Infection
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

2. 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 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,

3. 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

4. Human Immunodeficiency Virus
RNA virus
Surface envelope proteins
Matrix proteins
Capsule proteins
RNA

5. Evolution of HIV infection

6. 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

7. 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

8. 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).

9. 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.

10. HIV 1st Generation Antibody test

11. 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

12. 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

13. 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

14. 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.
The anti-HIV IgM antibody would attach to the antigens. Now the thing about the IgM antibody is that it is like many keys attached to each other. In other words there are multiple antigen binding sites. This is very different from IgG that has just 1 antigen binding site. So what the scientist figured out how to do was to stick the enzyme and a HIV antigen together. So this antigen-enzyme conjugate will stick onto the other binding sites of the IgM antibody that has stuck onto the antigens in the microwell. The enzyme on the antigen then converts the substrate color thus enabling detection. Since the IgM antibody is ‘sandwiched’ between 2 antigens, the name ‘sandwich ELISA’ was coined.

15. 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.
Not many people agree with this nomenclature because technically, it is identical to the 3rd generation ELISA test. Basically the 4th generation ELISA test is putting 2 different tests (i.e 3rd generation ELISA and P24 antigen test) onto the same test strip. Because the P24 antigen is produced even earlier than the IgM antibody, this reduces the testing window period even further

17. 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.

18. 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 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

19. 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
partial seroconversion during acute HIV infection
advanced HIV infection with decreased titers of p24 antibodies
infection with HIV-2.
Cross-reacting alloantibodies from pregnancy
Autoantibodies (collagen-vascular diseases, autoimmune diseases, and malignancy)
Receipt of an experimental HIV-1 vaccine
Influenza vaccination

20. HIV Immunofluorescent assay
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.

21. 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

22. 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

23. 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.

24. 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

25. 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.

26. Case 1 30 year old bisexual man comes into clinic
He has had 15 lifetime partners, never been HIV-tested
What test is appropriate?

27. 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?

28. 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?

29. Case 4 17 year old girl had a rapid HIV test that was positive
She comes to clinic for testing
What test is appropriate?

30. Case 5 17 year old girl had a rapid HIV test that was positive
The ELISA test was indeterminate
What test is appropriate?

31. 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?

32. Case 7 6 week baby had an HIV-infected mother
The mother receive treatment during pregnancy
What test is appropriate for the baby?

33. Thank You