Immunologic Methods in Diagnosis of HIV Infection. Tehran Medical Sciences Branch, Islamic Azad

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2 Immunologic Methods in Diagnosis of HIV Infection M Parsania, Ph.D. Tehran Medical Sciences Branch, Islamic Azad University

stranded d (+) RNA viruses that replicate

members that are oncogenic, are associated

3 Retroviridae Retroviruses (family Retroviridae) id ) are enveloped, single stranded d (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase. This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorders, and cause degenerative and neurological syndromes.

4 Retrovirus Classification Family: Retroviridae Genus Features Examples 1. Alpharetrovirus Simple, Onco Avian leucosis virus, RSV 2. Betaretrovirus Simple, Mouse Mammary Tumor Onco Virus 3. Gammaretrovirus Simple, Onco Murine leukemia virus (Moloney, Harvey) 4. Deltaretrovirus Complex, Onco 5. Epsilonretrovirus Complex, Onco Bovine Leukemia, Human T Cell Leukemia (HTLV) Walleye Dermal Sarcoma 6. Lentivirus Complex HIV, Visna, EIAV 7. Spumavirus Complex Simian Foamy Virus

5 RNA virus, 120nm in diameter Envelope gp160; gp120 & gp41 Family : Retroviridae Subfamily : Lentivirus Icosahedral symmetry Nucleocapsid Outer matrix protein (p17) Mj Major capsid protein ti (p24) Nuclear protein (p7) Diploid idrna with ihseveral copies of reverse transcriptase

6 HIV Structure surface transmembrane matrix protein capsid protein nucleocapsid protein RT Integrase protease

7 HIV Genome EARLY Accessory Genes LATE tat trans activator of transcription nef negative regulatory factor vif viral linfectivity it factor vpr viral protein R rev regulator of viral protein expression vpu viral protein U vpx HIV nucleotides (HIV 1): env gp160 (gp120:outer membrane part, gp41: transmembrane part) gag core proteins p55, p17 and p24 pol p66 (protease), p31,p51 (integrase/endonuclease)

8 Viral Replication Methods of transmission: Sexual transmission, presence of STD increases likelihood of transmission. Sharing contaminated needles (IV drug users). Transplantation of infected tissues or organs. Exposure to infected blood or bloodproducts products. Use of contaminated clotting factors by hemophiliacs. Motherto to fetus, perinatal transmission variable, dependent on viral load and mother s CD 4 count.

9 Viral Replication Firststep, step, HIVattaches to susceptible host cell. Site of attachment is the CD4 antigen found on a variety of cells helper T cells macrophages monocytes B cells microglial brain cells intestinal cells T cells infected later on.

10 HIV Life History CCR5 the co receptor HIV chemokine CD4 CCR5 CD4 CCR5 CD4 Mutant CCR5 macrophage

11 Virion interaction with CD4 receptor and CXCR4 co receptor

12 Early Phase HIV Infection In early phase HIV infection, initial viruses are M tropic. Their envelope glycoprotein gp120 is able to bind to CD4 molecules l and chemokine receptors called CCR5 found on macrophages

13 Late Phase HIV Infection In late phase HIV infection, most of the viruses are T tropic, having gp120 capable of binding to CD4 and CXCR4 found on T4 lymphocytes.

14 Retrovirus life cycle:

15 HIV and AIDS The cellular and immunological picture - The course of the disease

16 HIV Diagnostic Testing

17 Purpose of HIV Testing To identify asymptomatic individuals To diagnose HIV infection in those who practice high risk behavior To prevent secondary transmission Donor screening for blood & tissue products For prophylaxis, Mdi Medical management, ttreatment t For epidemiological surveillance To diagnose clinically suspected cases

18 Laboratory diagnosis of HIV infection Direct demonstration of infective agent - Virus isolation- virus culture - Antigen detection- P24 detection - viral nucleic acid detection- PCR Indirect demonstration of infective agent - Anti -HIV antibody detection

19 Initial and Supplemental HIV Tests Initial Test - Enzyme Immunoassay (EIA) - Chemiluminescent i Immunoassay (CIA) Supplemental Tests - Western blot - Indirect Immunofluorescence Assay (IFA) - Qualitative HIV-1 RNA

20 Spectrum of Immunologic Methods in Diagnosis of HIV Infection HIV diagnosis (Antibody/Antigen testing) Enzyme Immunoassays (EIAs) Rapid tests Western blot t(wb) Early diagnosis in infants p24 Initiation and monitoring of ART CD4 count

21 Enzyme Immunoassays (EIAs) Quantitative assay to measure HIV antibodies Most detect antibodies to HIV 1 and HIV 2 Antigens coated in microwells HIV Antigen / Antibody reaction is detected dby color change I t it f l fl t t f tib d t Intensity of color reflects amount of antibody present serum

22 After several incubation and wash steps, a color reaction occurs if HIV antibody is present An automated reader gives a measurement of optical density (presence of color) for each well

23 Generation of EIA Tests First Second Third *Fourth *Not US FDA approved as of 10/1/09 Uses crude viral Uses recombinant HIV Detects t IgM and IgG Detects t HIV antibodies lysate antigens or peptides in Sandwich EIA and p24 antigen

24 False Negatives and Positives False negative results may result from: Window period Agammaglobulinemia Seroreversion (loss of HIV antibodies due to extreme immune system destruction) in late disease Technical error Type N or O strains, or HIV 2 False positive results may result from: Autoantibodies e.g., SLE vaccines Technical error 24

25 Common HIV 1 or HIV 1/2 Diagnostic Algorithm

26 APHL/CDC HIV 1/2 Diagnostic Algorithm Template

27 Rapid tests Qualitative assay to detect HIV antibodies Most detect HIV 1 and HIV 2 As reliable as EIAs Advantages: 1. Quick 2. Easy to perform 3. No sophisticated instruments are required 4. Can be done on single sample Disadvantages: 1. Costly 2. Tedious if large no. samples have to be tested at one time

28 The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. Tests Based on Immunochromatography h Lateral Flow Devices Determine Hema Strip OraQuick Unigold The Control HIV Antigen Sample pad Specimen Flow

29 Body Fluids Used for HIV Rapid Testing Serum Plasma Whole blood Oral fluids

30 Reading Results Positive HIV-1/2 Reactive Control Positive Negative Read results in minutes

31 4 th Generation Immunochromatograpic test t for detection ti of HIV Ag/Ab Control bar (anti-hiv) p24 bar (avidin) Ab bar (rhiv Ag + synt peptides) Conjugate pad (Selenium colloid-hiv Ag/ Biotinylated anti-p24+ Selenium colloid anti-p24) B

32 4 th Generation Immunochromatograpic test t for detection ti of HIV Ag/Ab Control bar (anti-hiv) p24 bar (avidin) B Ab bar (rhiv Ag + synt peptides) B Conjugate pad (Selenium colloid-hiv Ag/ Biotinylated anti-p24+ Selenium colloid anti-p24) B Patient sample

33 Determine HIV-1/2 Ag/Ab Combo Control bar Selenium colloid Conjugate pad p24 Antigen bar Antibody bar

34 How Particle Agglutination Works Anti HIV antibodies bind to the antigen coated latex particles. Antigen Antibody

35 Proposed HIV Point of Care Algorithm HIV Diagnostic Algorithm (Rapid Test) Two Rapid Tests (A1/A2) Performed in Sequence on Blood or Oral Fluid (A1 and A2 must be different rapid tests) A1 [HIV-1 or HIV-1/2 rapid test (Blood or oral fluid)] A1+ A2 [HIV-1 or HIV 1/2 rapid test from a different manufacturer (blood)] A1- Negative for HIV-1 and dhiv2 HIV-2 antibodies* * A1+ A2+ Presumptive positive for HIV-1 or HIV-2 antibodies; requires medical follow-up for further evaluation and testing A1+ A2- Inconclusive rapid test result; requires additional testing *If using an HIV-1 only rapid test, Negative for HIV-1 antibodies only

36 Western Blot / Line Immunoassays Used as supplemental test for confirmation (only difficult cases) Detects antibodies to specific HIV antigens on cellulose strip

37 Confirmatory Tests Western Blot, Line immunoassay WB use antigens from whole virus lysates electrophoretically transferred to a membrane LIA use recombinant or synthetic HIV antigens mechanically applied on to support membrane Presence or absence of bands is scored Highly specific, Labor intensive, expensive WHO criteria- presence of at least 2 envelope bands (gp120, gp160,gp41) gp41)

38 HIV-1 Western Blot Antigens p = protein gp = glycoprotein Number = molecular weight

39 Components Used in HIV-1 Western Blot HIV Western blot Strip Color Reagent Y Y Y Enzyme Detector Antihuman IgG Antibodies Y Y Y Y YY Human HIV Antibodies (from patient serum) HIV Antigens (on Western blot)

40 HIV-1 and HIV-2 Gene Products & Western Blot

41 Interpretive Criteria for HIV-1 Western Blot Positive Control Negative Indeterminate Positive No bands: One or more bands present Not meeting positive criteria At least two of the Following bands: p24, gp41, gp120/160

42 Indirect immunofluorescence Can be used to detect both virus and antibody to it. Antibody detected by testing patient serum against antigen applied to a slide, incubated, washed hdand a fluorescent antibody added. Virus is detected by fixing patient cells to slide, incubating with antibody

43 Direct Methods of HIV diagnosis: p24 antigen detection EIA for detection of p24 antigen in serum, plasma, CSF or cell culture Can detect infection in window period, in late stage of disease,and in newborns To monitor response to anti-retroviral therapy To monitor disease progression Sensitivity is limited (only 69% in patients with AIDS and low in neonates < 1 month old) Negative test does not rule out HIV infection

44 Window Period Period between infection and first reliable detection of HIV by lab test. Window period varies by test and by individual. The majority of infected individuals are positive by ELISA, antigen, and/or DNA/RNA tests by 6 8 weeks after infection. 44

45 typical primary HIV 1 infection symptoms HIV proviral DNA symptoms window period HIV antibodies HIV viral load HIV-1 p24 antigen / infection weeks years Time following infection

46 Minimum Time from Infection to First Dt Detection ti of HIV 1 Markers KEY: TIME PERIOD BEFORE DETECTION TIME WHEN HIV DETECTION BY TEST IS POSSIBLE ELISA p24 DNA PCR RNA (VL) Infection Detection of HIV: time (in days) after infection 46

47 Detection of HIV by Diagnostic Tests Symptoms p24 Antigen HIV RNA HIV Enzyme Immune Assay (EIA)* Western blot Weeks Since Infection *4 th generation, Ag/Ab Combo EIA *3 rd generation, IgM-sensitive EIA *2 nd generation EIA *viral lysate EIA

48 Management of HIV Goal is undetectable (<48 copies/ml) HIV viral load (Assays vary on limit of detection from <75 to <20 copies/ml) Monitor the HIV viral load : 3 months once goal is achieved CD4 counts can be repeated : 6 12 months if viral lload controlled

therapy Manual and automated t methods Issues: Requires high level of

49 CD4 T Lymphocyte CD4 T lymphocyte counts used for: Determining clinical prognosis Assessing criteria for antiretroviral therapy Monitoring therapy Manual and automated t methods Issues: Requires high level of technical skill for test performance and interpretation Properly maintained equipment

50 CD4 Cell Counts CD4 cell count measures the number of CD4 cells per cubic milliliter of blood. The CD4 count is a measure of the degree of immunocompromise and stage of HIV disease progression. The CD4 count is an important test for deciding whether ARV therapy is required and for monitoring the recovery of the immune system under treatment. 50

Retroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses. members that are oncogenic, are associated

Retroviridae Retroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase. This large and diverse family includes