L02 Chapter 24 Laboratory Diagnosis of HIV Infection PDF

6/27/2024 LABORATORY DIAGNOSIS OF HIV INFECTION C H A P T E R 2 4 PREAMBLE PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is re...

6/27/2024 LABORATORY DIAGNOSIS OF HIV INFECTION C H A P T E R 2 4 PREAMBLE PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  6/27/2024 CHAPTER OVERVIEW Introduction Modes of HIV transmission Characteristics of HIV HIV and the immune system Clinical course of HIV infection Treatment and prevention Laboratory testing for HIV Screening and diagnosis Disease monitoring Testing of infants HUMAN IMMUNODEFICIENCY VIRUS (HIV) Cause of AIDS (acquired immunodeficiency syndrome) HIV-1 Cause of most HIV infections worldwide Four groups (M, O, N, P) Nine subtypes in group M (A, B, C, D, F, G, H, J, K) HIV-2 Originated in West Africa Causes fewer cases 2  6/27/2024 MODES OF HIV TRANSMISSION Contact with blood or Sexual contact Perinatal other body fluids Intimate contact IV drug use Mother to child: involving exchange Recipients of before, during, or of body fluids transfusions, other after birth (through Responsible for blood products breast milk) majority of cases Occupational injury (direct needlestick or mucous membrane exposure) HIV Virus has been isolated from body fluids Not infective urine saliva tears MODES OF HIV Infective TRANSMISSION blood Semen & vaginal secretions Synovial, Pleural, Peritoneal and Pericardial CSF breast milk 3  6/27/2024 CHARACTERISTICS OF HIV Retrovirus Contains two copies of ssRNA Reverse transcriptase transcribes the viral RNA into DNA. Surrounded by a protein coat (capsid) Outer envelope of glycoproteins embedded in a lipid bilayer STRUCTURE OF HIV VIRION 4  6/27/2024 HIV GENOME R E P L I C AT I O N O F H I V Attachment of HIV to host cell Main target: CD4 Th Coreceptor required Fusion and uncoating Reverse transcriptase converts viral RNA into complementary DNA (cDNA). 5  6/27/2024 R E P L I C AT I O N O F H I V ( C O N T I N U E D ) cDNA is integrated into the host genome as a provirus. Viral DNA is transcribed into viral RNA and proteins. Viral particles are assembled. Intact virions bud out of host cell membrane. IMMUNE RESPONSES TO HIV Humoral antibody Cell-mediated Innate defenses production immunity NK cells mediate Antibodies are T cells produce cytolysis of HIV- detected by 6 weeks cytokines with infected cells. after infection. antiviral activity. Dendritic cells Antibodies CTLs destroy HIV- stimulate release of produced later may infected host cells. cytokines that have prevent HIV from antiviral effects. infecting host cells and participate in ADCC. 6  6/27/2024 Genetic mutations rapidly occur, generating new viral mutants with altered antigens. HIV can downregulate expression of MHC-I molecules on infected host cells. HIV ESCAPE FROM HIV can survive as a latent provirus for prolonged IMMUNE periods. RESPONSES As a result, HIV persists and destroys the immune system. CD4 T cells are the prime targets of destruction, resulting in reduced effectiveness of antibody and cell-mediated immune responses. CLINICAL COURSE OF HIV INFECTION Primary infection 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 latency Absence of clinical symptoms Decrease in viremia and increase in CD4 T-cell number AIDS Resurgence of viremia and decrease in CD4 T-cell number Profound immunosuppression, with appearance of life-threatening opportunistic infections and malignancies 7  6/27/2024 Drugs that block various steps of the HIV replication cycle Nucleoside analog reverse transcriptase inhibitors Nonnucleoside reverse transcriptase inhibitors Protease inhibitors Integrase inhibitors Fusion inhibitors CCR5 antagonists ANTIRETROVIRAL Post attachment inhibitors THERAPY (ART) Are most effective when used in combination. Has significantly improved morbidity and mortality of HIV-infected persons and has reduced the rate of perinatal transmission. PREVENTION OF HIV TRANSMISSION Screening blood/organ donors for HIV Education of the general public on HIV transmission, safety measures Precautions for health-care workers Vaccine—being researched 8  6/27/2024 L A B O R ATO R Y T E S T I N G F O R H I V Screening Monitoring Testing Screening and Disease Testing of infants diagnosis monitoring Previous algorithm CD4 T-cell count and test methods HIV viral load Current algorithm Drug resistance and test methods testing PREVIOUS CDC TESTING ALGORITHM Screen for HIV-1/HIV-2 antibodies by ELISA or rapid EIA. Confirm positive test results by repeating ELISA, followed by Western blot. 9  6/27/2024 Also known as Immunoblotting Tests for a specific protein within a protein mixture. The Western blot test is performed after gel- electrophoresis has separated protein It uses antibodies to identify specific proteins. WESTERN The separated proteins are transferred (or blotted) BLOT TEST onto nitrocellulose or nylon membranes and identified by specific antibodies that are tagged by a secondary protein. WESTERN BLOT I N TE R PR E TAT I O N Interpretation: No bands = negative Positives are harder to interpret CDC says must have antibody against two of three of the following bands: P24 gp41 gp 120/160 These two bands are so close can’t get good separation to be considered positive 10  6/27/2024 CURRENT CDC TESTING ALGORITHM PRINCIPLE OF FOURTH-GENERATION HIV-1ANTIBODY/HIV-2A NTIBODY/P 24 ANTIGEN COMBINATION IMMUNOASSAY Incubate patient serum with solid phase onto which HIV-1 antigens, HIV-2 antigens, and antibody to HIV-1 p24 have been bound. 11  6/27/2024 PRINCIPLE OF FOURTH-GENERATION HIV-1ANTIBODY/HIV-2ANTIBODY/P24 ANTIGEN COMBINATION IMMUNOASSAY Following incubation, HIV-1 or HIV-2 antibodies in the patient sample will bind to their respective antigens. HIV-1 p24 antigen in the patient sample will bind to anti-p24 on solid phase. Wash, then add conjugate consisting of labeled anti-p24 and labeled HIV-1/HIV-2 antigens. FOURTH-GENERATION HIV-1 ANTIBODY/HIV-2 ANTIBODY/P24 ANTIGEN COMBINATION ASSAY R E S U LT S Following an incubation, wash, and addition of trigger solution or substrate/stop solution, relative light units or optical absorbance are measured. Confirm positive results with rapid EIA. 12  6/27/2024 HIV DISEASE MONITORING Peripheral Quantitative Drug blood CD4 T- viral load resistance and cell counts assays tropism testing C D 4 T- C E L L E N U M E R AT I O N CD4 T-cell numbers are the best indicator of immune function in HIV- infected individuals. Incubate peripheral blood with fluorescent-labeled anti-CD4; analyze results by flow cytometry. In untreated patients, CD4 T-cell number declines progressively, and CD4 T:CD8 T-cell ratio is less than 1:1. CD4 T-cell count of less than 200/μL indicates stage 3 HIV infection (AIDS). A significant decline in CD4 T-cell count over time may indicate a need to change ART or administer prophylactic therapy for certain infections. 13  6/27/2024 Measure amount of HIV RNA circulating in patient plasma Methods: qPCR bDNA HIV RNA detectable about 11 days after infection. Q UAN TITAT IV E Successful therapy with ART results in a decline in the viral load to an VIRAL LOAD undetectable level. ASSAYS Patients with a persistently increased viral load should undergo drug resistance testing and may need a possible change in ART. C D 4 T- C E L L N U M B E R S A N D P L A S M A VIREMIA DURING HIV INFECTION 14  6/27/2024 D R U G R E S I S TA N C E T E S T I N G Genotype resistance assays Performed in clinical laboratory settings HIV reverse transcriptase and protease genes from RNA in patient plasma are amplified by RT-PCR. Products are sequenced and analyzed with software for mutations. Results are reported as: Resistance Possible resistance No evidence of resistance D R U G R E S I S TA N C E T E ST I N G (CONTINUED) Phenotype resistance assays Tropism testing Determine ability of HIV Genotypic or phenotypic from clinical samples to assays are performed to grow in the presence of determine if the patient has antiretroviral drugs. virus that will bind to the Involve sophisticated CCR5 co-receptor and be technologies only responsive to CCR5 performed by specialized antagonists. reference laboratories. 15  6/27/2024 T E ST IN G O F IN FA N T S YO UN G E R THAN 18 MONTHS Maternal antibodies in All pregnant women infant serum can Molecular methods should be tested for complicate serological are used for diagnosis. HIV infection. test results. Qualitative HIV-1 DNA Serological testing at PCR using infant’s 12 to 18 months of peripheral blood age may be used to mononuclear cells is confirm the diagnosis. the preferred method. SUMMARY HIV-1 is the cause of the majority of AIDS cases throughout the world. Transmission of HIV occurs: By intimate sexual contact By contact with contaminated blood or body fluids Perinatally HIV is a retrovirus with three structural genes: gag pol env 16  6/27/2024 The primary targets of HIV infection are CD4 T cells and macrophages; CD4 serves as the main receptor for entry of the virus into the host cells. Although the body mounts innate defenses and produces specific antibodies and cytotoxic T-cell responses against HIV, the virus has developed SUMMARY several escape mechanisms that allow it to (CONTINUED_1) persist. There are three phases in the natural course of HIV infection: Primary infection, Latency and AIDS Treatment with ART is recommended for all HIV-infected persons and has resulted in delayed disease progression, decreased mortality, and reduced perinatal transmission. The current CDC testing algorithm for HIV infection consists of screening by a fourth-generation immunoassay that detects HIV-1 and HIV-2 antibodies and p24 antigen. SUMMARY Positive results are confirmed with a rapid EIA that (CONTINUED_2) distinguishes between HIV-1 & HIV-2. Discrepant results are resolved by nucleic acid testing. 17  6/27/2024 The current algorithm is more accurate and faster than the previous testing scheme, which involved screening with an ELISA and confirming positive results with a Western blot. Patients with HIV infection are routinely monitored by performing CD4 T-cell enumeration and viral load testing. SUMMARY (CONTINUED_3) Molecular methods play an important role in resolving discrepant screening results, monitoring patient viral load during ART, detecting antiviral drug resistance, and diagnosing infections in infants younger than 18 months of age. P O STA M B L E READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 18