Serology Exam #2: Lecture #7

HIV

Human Immunodeficiency Virus (HIV): Type of virus, inner core, reverse transcriptase, capside

• Retrovirus

• Contains inner core with two copies of ssRNA

• Reverse transcriptase transcribes the viral RNA to DNA

• Surrounded by protein coat (capsid)

Human Immunodeficiency Virus (HIV); Outer envelope & antigen proteins

• Outer envelope of glycoproteins embedded in lipid bilayer

• Antigen proteins: p (protein) & gp (glycoprotein)

HIV Genome: gag vs pol vs env

• gag = codes for nucleocapsid & core proteins

• pol = codes for viral enzyme

• env = codes for envelope proteins

HIV modes of transmission (3)

sexual contact, contact with blood or other bodily fluids, & perinatal

HIV Replication: Attachment of HIV, main target cell, coreceptor

• Attachment of HIV to host cell: gp120 binds to CD4 on cells surface

• Main target: CD4+ T cells (macro, mono, DCs, langerhans cells, microglial brain cells)

• Coreceptor required (chemokines): CXCR4 (T cells) & CCR5 (macrophages)

HIV Replication: DNA into host, viral DNA transcribed, intact virions

• DNA integrated into host genome as provirus

• viral DNA transcribed into viral RNA & proteins

• intact virions bud out of host cell membrane

HIV Effects on the Immune System:

• HIV can downregulate expression of MHC-I molecules on infected host cells

• HIV can survive as a latent provirus for prolonged periods

• HIV persists and destroys the immune system

Clinical Course of HIV Infection: Primary infection (infection severity, symptoms, high level of, decrease in)

• Acute, early infection

• May be asymptomatic or have a flu-like syndrome that resolves

• High level of viremia and decrease in CD4 T-cell number

Clinical Course of HIV Infection: Clinical latency (symptoms, decrease in, increase in, LTNP)

• Absence of clinical symptoms

• Decrease in viremia and increase in CD4 T-cell number

• Long-term nonprogressors (LTNP) – patients that remain asymptomatic for more than 10 years without antiviral therapy

  • normal or mildly decreased CD4+ counts, and low viral load

Clinical Course of HIV Infection: AIDS (resurgence, symptoms/manifestations)

• Resurgence of viremia and decrease in CD4 T-cell number

• Profound immunosuppression, with appearance of life-threatening opportunistic infections and malignancies

HIV Staging: Stages & Stage 0 (test results, reclassified)

• 5 stages (0, 1, 2, 3, 4)

• Stage 0: Patients with early HIV infection

  • Have 1 positive HIV test, followed by a neg or indeterminate test within 6 months

  • Reclassified into one of the other categories 180 days or more after initial diagnosis

HIV Staging: Stages 1-3, Unknown stage, Stage 3

• Stages 1, 2, and 3 based on CD4 T-cell counts (decreases with severity)

• Unknown stage is used if CD4 data is missing

• Stage 3 is also indicated if any opportunistic illnesses are present

Laboratory Testing for HIV (3)

• Nucleic acid testing (NAT)

• HIV antibody detection

• HIV antigen detection

Previous Testing Algorithm for HIV: Screening tests (2), confirm pos test, pos for western blot

• Screen for HIV-1/HIV-2 antibodies: ELISA & Rapid EIA

• confirm pos test: repeat ELISA followed by western blot

• Pos must have at least 2 of 3 bands present: p24, gp41, and gp120/gp160

Current Testing algorithm

• combination immunoassay to detect HIV-1, HIV-2 antibodies, and p24 antigen

• HIV-1/2 antigen/antibody combination immunoassay (+) → HIV-1/2 antibody differentiation immunoassay → if (-) for both → HIV-1 NAT

Fourth-Generation Combination ELISA: Type of test & Principle (7)

• Combination, sandwich ELISA

1. Recombinant HIV-1 and HIV-2 antigens, AND monoclonal antibody to HIV-1 p24 attached to solid phase

2. incubate with patient serum

3. wash

4. Add conjugate containing enzyme-labeled anti-p24 and HIV-1/HIV-2 antigens

5. Incubate, then wash

6. Add trigger solution or stop solution

7. Measure optical absorbance

Tests for Disease Monitoring: Peripheral blood & Drug resistance

• Peripheral blood CD4 T-cell counts: Flow cytometry

• Drug resistance testing: Genotype and phenotype resistance assays

CD4 T-Cell Enumeration: Indicator, Incubate, in untreated patients, CD4 T cell count

• Best indicator of immune function in HIV-infected individuals

• Incubate peripheral blood with fluorescent-labeled anti-CD4

• In untreated patients, CD4 T-cell number declines progressively

• CD4 T-cell count of less than 200/mcL indicates stage 3 infection (AIDS)

Drug Resistance Testing - Genotype: Assays (detect, type of PCR), Susceptibility (report as)

• Genotype resistance assays

  • Detect mutations in reverse transcriptase and protease genes from RNA in patient plasma

  • Amplified RT-PCR

  • Nucleotide sequenced products entered into database & analyzed with software for mutations

• Predictable susceptibility

• Report as: Resistance, Possible resistance, No evidence of resistance

Drug Resistance Testing - Phenotype: Assay (determines, direct measure), recombinant virus, where performed

• Phenotype resistance assays

• Determine ability of HIV from clinical samples to grow in presence of antiretroviral drugs

• Direct measure of drug susceptibility

• Create recombinant virus from patient plasma and laboratory reference strain using mammalian cells

• Only performed by specialized reference laboratories

Testing in Infants: Age, antibodies, test for diagnosis, serologic testing

• < 18 months of age

• Maternal antibodies in infant serum can complicate serologic test results

• Qualitative HIV-1 DNA PCR (preferred method)

• Serologic testing at 12 to 18 months of age may be used to confirm diagnosis