hiv

Laboratory Diagnosis of HIV Infection Overview

• Course: MLS 227

• Chapter: 24

Human Immunodeficiency Virus (HIV)

• Definition: Cause of AIDS (Acquired Immunodeficiency Syndrome)

• Types of HIV:

  • HIV-1:

  • Responsible for most HIV infections globally.

  • Divided into four groups: M, O, N, P.

  • Group M contains nine subtypes: A, B, C, D, F, G, H, J, K.

  • HIV-2:

  • Originated in West Africa.

  • Accounts for fewer cases than HIV-1.

Modes of HIV Transmission

• Sexual Contact:

  • Involves intimate contact with exchange of body fluids.

  • Responsible for the majority of HIV infection cases.

• Contact with Blood or Other Body Fluids:

  • Includes IV drug use, transfusions, or occupational injuries (e.g., needle stick, mucous membrane exposure).

• Perinatal Transmission:

  • Transmission from mother to child before, during, or after birth, including through breast milk.

Characteristics of HIV

• Classification: Retrovirus.

• Composition:

  • Contains two copies of single-stranded RNA (ssRNA).

  • Reverse Transcriptase: Enzyme that transcribes viral RNA into DNA.

• Structure:

  • Surrounded by a protein coat called a capsid.

  • Maximally characterized by an outer envelope of glycoproteins embedded in a lipid bilayer.

Structure of HIV Virion

• Various components:

  • Docking Glycoprotein (gp120)

  • Transmembrane Glycoprotein (gp41)

  • Integrase

  • Matrix Protein (p17)

  • Reverse Transcriptase

  • Capsid Protein (p24)

  • Viral RNA

  • Phospholipid Envelope

HIV Genome

• Structural Genes:

  • 5' LTR

  • gag: Codes for structural proteins.

  • pol: Codes for viral enzymes (reverse transcriptase, integrase).

  • env: Codes for viral envelope proteins (gp160 precursor leading to gp120 and gp41).

• Regulatory Genes:

  • vif, vpr, tat, rev, vpu, nef

  • Influence virus replication and immune evasion.

• 3' LTR

Replication of HIV

• Steps in the replication cycle:

  1. Attachment: HIV attaches to host cells, primarily targeting CD4 T cells.

  2. Fusion and Uncoating: The virus enters the cell.

  3. Reverse Transcription: Reverse transcriptase converts viral RNA into complementary DNA (cDNA).

  4. Integration of cDNA: cDNA is integrated into the host genome as a provirus.

  5. Transcription and Translation: Viral DNA is transcribed into viral RNA and proteins.

  6. Assembly: Viral particles are assembled.

  7. Budding: Intact virions bud out from the host cell membrane.

Immune Responses to HIV

• Innate Defenses:

  • Natural Killer (NK) Cells: Mediate cytolysis of HIV-infected cells.

  • Dendritic Cells: Stimulate release of cytokines with antiviral effects.

• Humoral Antibody Production:

  • Antibodies detectable typically six weeks after infection.

  • Later produced antibodies may prevent HIV from infecting new host cells.

• Cell-Mediated Immunity:

  • T Cells: Produce antiviral cytokines.

  • Cytotoxic T Cells: Destroy HIV-infected cells.

HIV Escape from Immune Responses

• Mechanisms of evasion include:

  • Genetic Mutations: Rapid alterations generate new viral mutants with altered antigens.

  • MHC-I Downregulation: Infected cells reduce MHC-I molecule expression.

  • Latent Provirus: HIV can remain hidden for extended periods.

• Result: Continuous persistence and devastation of the immune system, especially CD4 T cells leading to impaired antibody and cell-mediated responses.

Clinical Course of HIV Infection

• Primary Infection:

  • Acute phase; may present asymptomatically or with flu-like symptoms.

  • High levels of viremia and decreased CD4 T-cell counts.

• Clinical Latency:

  • No clinical symptoms; decrease in viremia and increase in CD4 T-cell counts.

• AIDS:

  • Resurgence of viremia with decreased CD4 T cells.

  • Severe immunosuppression, leading to opportunistic infections and malignancies.

Antiretroviral Therapy (ART)

• Purpose: Blocks various stages of HIV replication.

• Effectiveness: Optimal when combined with other drugs.

• Impact: Significantly improves morbidity and mortality among HIV-infected individuals and reduces perinatal transmission rates.

Prevention of HIV Transmission

• Strategies include:

  • Screening blood and organ donors for HIV.

  • Public Education on HIV transmission and safety measures.

  • Health Care Workers: Implementation of standard precautions.

  • Vaccine Research: Current ongoing studies.

Superinfection Dynamics

• Initial Infection vs. Superinfection:

  • Superinfection: Refers to an individual acquiring a new strain of HIV after initial infection.

  • Possible implications for treatment and viral dynamics.

Laboratory Testing for HIV

• Screening and Diagnosis:

  • Overview of previous and current testing algorithms.

  • Importance of disease monitoring through metrics like CD4 T-cell count, viral load, and drug resistance testing.

• Previous CDC Testing Algorithm:

  • Screening: Use of ELISA or rapid EIA for antibodies.

  • Confirmation: Positive results reviewed with ELISA followed by Western blot.

Current CDC Testing Algorithm

• Initial Testing: Combination immunoassay for HIV-1/2 antigen/antibody.

• Interpretation:

  • Multiple follow-up assays to distinguish between HIV-1 and HIV-2 antibodies.

  • Utilizes nucleic acid testing (NAT) for confirmation of acute infections or indeterminate results.

HIV Disease Monitoring

• Metrics include:

  • Peripheral Blood CD4 T-cell Counts: Best indicator of immune function in HIV-infected persons.

  • Quantitative Viral Load Assays: Measures circulating HIV RNA in plasma.

  • Drug Resistance and Tropism Testing: Evaluates resistance to treatments and co-receptor usage.

CD4 T-Cell Enumeration

• Methods:

  • Incubation with fluorescent-labeled anti-CD4 antibodies analyzed via flow cytometry.

• Clinical Significance:

  • Declines in CD4 counts indicate the progression of HIV infection.

  • Counts below 200/μL signify stage 3 HIV infection (AIDS).

Quantitative Viral Load Assays

• Purpose: Measure circulating HIV RNA in plasma.

• Techniques:

  • Quantitative Real-Time PCR (qPCR)

  • Branched Chain DNA (bDNA)

• Clinical Relevance: Viral loads can indicate urgency for ART modification; successful therapy results in viral loads dropping to undetectable levels.

Drug Resistance Testing

1. Genotype Resistance Assays:

   • Amplifies HIV reverse transcriptase and protease genes for sequencing and mutation analysis.

   • Results: Show resistance levels (e.g., resistant, possible resistance).

2. Phenotype Resistance Assays:

   • Evaluate virus growth in the presence of antiretroviral drugs; performed in specialized labs.

3. Tropism Testing:

   • Determining if virus will bind to the CCR5 co-receptor, aiding in treatment decisions.

Testing of Infants Younger Than 18 Months

• Importance of maternal testing during pregnancy.

• Challenges posed by maternal antibodies in serological testing.

• Preferred Diagnostic Method: Qualitative HIV-1 DNA PCR using infant’s peripheral blood.

• Follow-up: Serological tests at 12-18 months to confirm infection.

• Role of Molecular Methods: Critical for managing treatment and monitoring viral load in infants.

Summary of HIV Infection

• HIV-1 is the leading cause of AIDS globally, transmitted through intimate contact, blood, and perinatal routes.

• HIV is categorized as a retrovirus, with major structural genes including gag, pol, and env, targeting CD4 T cells and macrophages.

• HIV has several mechanisms for evasion of the immune response, which complicates treatment.

• Clinical progression of the infection includes three main phases: primary, latency, and AIDS.

• ART improves health outcomes and reduces rates of transmission.

Summary of Current Testing and Monitoring Techniques

• The CDC's current HIV testing protocol includes advanced immunoassays and confirmed testing methods that significantly enhance diagnostic accuracy and patient management. This transitions from previous methods that relied heavily on ELISA and Western blot.

• Regular monitoring through CD4 T-cell counts and viral load testing are vital for managing HIV infection.