2. Laboratory Testing for HIV Diagnosis
• Antibody Assays:
– Rapid tests
– ELISA
• Confirmatory Assays
– Western Blot
– Indirect Immunofluorescent Antibody Assay (IFA)
• Nucleic Acid-based Test (Virologic tests)
– Assays to detect HIV DNA or RNA (PCR)
– Other tests:
– 1. Antigen detection test (p24 antigen)
– 2. Viral isolation
3. Antibody Tests
• Antibody tests for diagnosing HIV include:
– Rapid tests
– Enzyme Linked Immunosorbent Assay (ELISA)
– Western Blot (confirmatory test if available)
– Indirect Immunofluorescent Antibody Assay (IFA)
• Effective 12 weeks after infection, as antibodies
appear 6 to 12 weeks following HIV infection in a
majority of patients
• Not suitable for use in infants under 18 months (use
PCR instead)
4. Nucleic Acid-Based Tests
• DNA/RNA PCR tests consists of:
– DNA Polymerase Chain Reaction (DNA PCR) and
– Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
• These tests are not routinely used for HIV diagnosis in
adults but rather useful in:
– Early diagnosis and infant diagnosis (<18 months)
– Situations when it is desirable to exclude serologic latency
period, such as artificial insemination and other tissue or
organ transplant procedures
5. National Testing Algorithm
• Government has approved the use of HIV Testing
Algorithm
• To support testing in the country government has
approved a combination of non-cold chain dependent
HIV rapid test kits to be used in the National Algorithm.
– The kits are Determine, UniGold and SD Bioline
6. Testing Algorithm
Perform first test (Assay 1 or A1)
Perform second test (A2)
Report HIV +
Immediately refer for ART (Test and Start)
A1+
A1−
Report HIV-negative
Repeat A1 and A2
A1+ A2+ A1+ A2−
A1− A2−
Report HIV-negative
A1+ A2+
A1+ A2−
Report HIV-inconclusive; retest in 14
days
7. Result Interpretation: >18months
• For Children greater than 18 months
– Means that this child is confirmed to be HIV
infected
– Such a child should be enrolled into PreART and
ART register (on commencement of ART) as
soon as possible.
8. Result Interpretation: <18months
• For Children less than 18 months
– Means that this child is an exposed infant. We do
not know if child is infected but such a child will
require extra care and early infant diagnosis
– Such a child should be enrolled into HIV exposed
infant care, sent for virologic testing (DBS) and
followed up as per national guideline.
9. Serologic Latency: Window Period
• HIV antibodies take time to multiply and show up on
antibody or antigen tests
• The window period is the period of serologic latency
between the time of infection and when initial detection of
HIV markers is possible by laboratory tests
– Influenced by the HIV marker, the assay and by individual patient
10. Serologic Latency: Window Period
• The majority of infected individuals test positive by
antibody, antigen and/or DNA/RNA test 6-8 weeks after
infection
• However, the window period can last as long as 3 to 6
months
– Especially true for patients with Hepatitis C co-infection (window
period can last up to 12 months)
EXPLAIN that there are various types of rapid tests, which will be covered in this session.
NOTE that the ELISA was the first test used, though it has drawbacks, particularly in developing countries. EXPLAIN the drawbacks of the ELISA test:
It is a complex diagnostic process that requires highly trained personnel, expensive equipment, and a high cost per test. Note that this high cost is not sustainable and will thus limit access to the test.
It is not suitable for point of care as testing is done in batches, meaning that test results cannot be made available on the same day.
ASK participants, “What does PCR mean?”
PCR = Polymerase Chain Reaction
EXPLAIN that both Rapid Tests and ELISA tests are used for HIV screening. The ELISA procedure is carried out to screen for HIV IgG antibodies in plasma or serum. Rapid tests are user-friendly.
Commonly used rapid antibody tests in South sudan:
Dot immunoassay (e.g., UniGold)
Chromatography (e.g. Determine)
The Western Blot and IFA are confirmatory assays. Western blot is the standard confirmatory test for HIV antibody assays. IFA employs HIV-infected cells (lymphocytes) fixed to the microscope slide. IFA has been used to confirm diagnosis in sera producing indeterminate results in Western blot.
EXPLAIN that double rapid tests are currently used instead of the Western blot test as HIV testing scales up.
EXPLAIN PCR tests.
EXPLAIN that virologic tests detect the virus or viral particle.
ASK participants: “What’s the difference between DNA and RNA PCR?”
RNA PCR is quantitative and shows actual copies of viruses (viral load), while DNA PCR is qualitative and is used in early infant diagnosis.
Before advancing to the next slide, ASK participants of the testing algorithm(s) are.
ADVANCE to the next slide to discuss the definitions and summarize the discussion.
NOTE to participants that there are several other tests. ASK participants to name them. RECORD their responses on a flip chart.
If possible, EXPLAIN that the name of the test indicates the methodology utilized. e.g. Determine uses chromatography
REFINFORCE for participants that because both antibody and antigen tests rely on antibodies, and antibodies take 6-12 weeks to show up on a test, HIV cannot be immediately detected through these tests. For that reason, there is a window period.
REMIND participants that this also means that someone can be infected and infectious to others even though the antibody test was negative.
EXPLAIN to participants that individuals can have different window periods.
ASK participants, “What causes these differences?” Possible response: assay used, nutritional status, agammaglobulinemia and anemia
EXPLAIN that during this period, an antibody test can cause a false negative reading. False-negative HIV tests can also be a result of:
Unexplained atypical host response
Agammaglobulinaemia
Seroreversion: Certain phase of the disease when B cell function is completely paralysed
Excessive antigen production mopping up all the antibodies formed to uncommon strains of HIV, such as HIV-1 Type N and O strains
Technical errors
