Laboratory Diagnosis of Human Immunodeficiency Virus

(HIV) Dr Mostafa Mahmoud, MD, Ph D,

Consultant MicrobiologistAssist. Prof. of Medical

Microbiology & Immunology

What is the immune system?

The immune system is the collection of cells, tissues and molecules that protects the body from numerous pathogenic microbes and toxins in our environment.

What are the types of immune system?2 main types:1- the Innate, or natural (Non-specific) immune system (fight microbes at site of entry) include: 1) physical epithelial barriers, 2) phagocytic leukocytes, 3)

dendritic cells, 4) a special type of lymphocyte called a natural killer (NK) cell, and 5) circulating plasma proteins.

2- the Acquired or adaptive (Ag-Specific) immune system:

-The second line of defense after innate -Naturally silent need activation by infection with

formation of memory cells.

-2 subdivisions of the adaptive or acquired the humoral (Antibodies- by B-lymphocytes) and the cellular (CMI by T-lymphocytes).

A. Humoral Immunity - By antibodies from B-lymphocytes- B-lymphocytes 10-20% of circulation lymphocytes. - (WBCS: Neutrophils, lymphocytes, Eosinophil,

Monocytes & basophils), - Also in bone marrow, spleen, LNs. - Different antibody specific to each antigen.- 5 classes of antibodies (Immunoglobulin; Igs) IgM (acute), IgG (chronic), IgA (secretions), IgD (receptors) IgE (hypersensitivity)

B-Cell-Mediated Immunity (CMI)- By T-lymphocyte populations that integrate in function.- Against viruses, fungi, intracellular bacteria, and tumor cells.- T-lymphocytes are Thymus-dependent present in spleen and LNs.

60% to 70% of circulating lymphocytes.- T-lymphocytes include subsets: Helper T-cells (CD4+) which help B-lymphocyte function by

several cytokines. Cytotoxic T-cells (CD8+) killing infected or tumor cells. Suppressor T-cells Memory T-cells

• What is AIDS ?= Acquired Immuno- Deficiency-Syndrome- It is the a disease syndrome

•.

• What is HIV ? = Human Immunodeficiency Virus

- It is the causative viral agent of ADIS- Enveloped ?? SS-RNA, Positive-Sense (2

segments)• HIV is a Retrovirus?• HIV-1, the most common worldwide• HIV-2, few areas in Africa.

HIV-Structure(Viral Antigens)

1- Gag= Group Antigen: (P24 (Capsid), P17 (Matrix), & P7 (Nucleocapsid).

2- Pol= polymerase and associated enzymes: P66 (reverse transcriptase -RT), P32 (integrase) , P9 (protease).

2- Env= Envelope glycoproteins e.g. GP 120, GP 41.

HIV infects (CD4+) carrying cells!!!The effects of HIV are due to it gp 120 Ag.

The CD4+ are the receptors of the virus.HIV affects the immune system & Brain??Why?They have CD4+Tmainly on T-helper cellsOther cells having CD4+ include: - Macrophages - Dendritic cells.- Monocytes - Microglial cells- Retinal cells - Colonic Mucosal

cells.

(GP 120 &41)

(PCR)

(GAG)

Diagnostic lab test for HIV infectionA- Tests to diagnose HIV infections:1- Screening2- ConfirmatoryB- Tests for follow up the disease:1- Viral load by Quantitative PCR 2- Th-cells count (CD4+)C- Tests for the complication of the disease:1- TB infection 2- HBV 3- HCV

4- Toxoplasma5- Liver function tests 6- UTIs7- Others.

A- Tests to diagnose HIV infections1- Screening tests These test are rapid in giving results however, they are not diagnostic and need confirmation by the confirmatory testsThe Screening tests include:i- ELISA tests (combined Ag-Ab Immunoassays) for detecting HIV-1 & HIV-2 antibodies and P24 Ag of HIV-1. or chemiluminescent immunoassay testing.ii- Rapid tests

i- ELISA or chemiluminescent immunoassay testing

Were previously for detection of HIV AntibodiesNow detection of P24 Antigen of HIV is included

(Combo ELISA).Even though, need confirmationThey are the tests approved and done in our hospitalsDone upon serum sample If initially reactive, repeat in triplicate, if positive send

for WB or PCR.

Screening test performed in 1- Premarital examination2- Blood Donors.3- Antenatal Care4- Pre-employment screening5- Before Surgical operations (Medico-legal)6- Follow up for hemodialysis patients.7- Others

Semi-Automated ELISA (separate washer, Incubator, Reader)ELISA washers (for semi-automated)

Stat Fax microplate washer Bio-Tek microplate washer

In 50-bed Hospitals of MOH

Manual ELISA readers

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

Fully Automated ELISA machines (dilution, pipetting, washing, incubation and reading)

Evolis (Bio-Rad) Eti- Max (Diasorin)

•Do not forget ELISA is Screening not Confirmatory

ELISA TestingCurrently Previously Detection of Antibodies and Antigens

Detection of Antibodies only

Early results within hours or days of infection by detecting Antigens

Results positive only after 4-8 weeks after infection

For HIV-1 & HIV-2 For HIV-1 onlyFull automation available Cumbersome

ii- Rapid tests Advantages Easy to use immediate and fast results (10-

20 minutes).Can be used at home or clinic. Inexpensive (1-2 $/test)Recently approved by MOHSome tests are FDA approvedDone upon whole blood or saliva No equipment, refrigeration, No electricity, No

multiple timing steps required.Ø Built in controls

Disadvantages of rapid tests.

Only detects Antibodies to HIV 1/2Not detecting AntigensPositive late after infection; after

seroconversion. Need confirmation for reactive tests.Subjective variability in result reading.

Rapid tests for HIV diagnosis

Home Rapid tests.

Add Sample

ConjugateControl

lineTest Line

IgG Antibodies HIV antibodies

Colloidal goldconjugated to HIV antigen

Anti-IgG/goldantibodies

HIV antigen

How Immunochromatography Works

Lab workers Health workers

Clinic Rapid Tests.

2- HIV Diagnostic Confirmatory tests

1- Differential Antibody tests (Western Blotting, WB).2- Indirect Immunofluorescene Assay (IFA). Not available in MOH3- Nucleic acid Amplification tests (NAT, PCR) done in most hospitals for screening blood donors and in RRL for confirmation of screening tests.4- Virus Isolation (not done for clinical diagnosis only in research purposes).

1- Differential Antibody tests (Western Blotting).

Differentiate antibodies to HIV-1 from HIV-2 and antibodies to specific antigens.

Time consumingMay give indeterminate resultsPerformed in Riyadh Regional Lab.

Western Blot for detection of various antibodies to various parts of the virus

When to ask for HIV infections?when they present with a febrile, “flu”-, or “mono”-like illness that is not otherwise explained:- • Those who present for HIV testing (AII) • Those who report a recent sexual or parenteral exposure with

a known HIV-infected partner or a partner of unknown HIV serostatus in the past 2 to 6 weeks (AII)

• Men who report having unsafe sexual practices with other men (AII)

• Those who report needle-sharing (AII) • Those who present with a newly diagnosed sexually

transmitted infection (AII) • Those who present with aseptic meningitis (AII) • Pregnant or breastfeeding patients (AII)

When acute HIV infection is suspected: • An HIV serologic screening test should be used

in conjunction with a plasma HIV RNA assay (AII); • Detection of HIV RNA or antigen in the absence

of HIV antibody is a preliminary positive result; HIV RNA testing to be repeated for confirmation of HIV RNA

• Both serologic and RNA testing should be repeated to exclude a false-positive result with <5,000 copies/mL HIV RNA detected in serological negative patient (AII).

CDC Recommended Laboratory HIV Testing Algorithm for Serum or Plasma Specimens

(WB)

(ELISA)

(PCR)

REASONS FOR FALSE-POSITIVE, FALSE-NEGATIVE, AND INDETERMINATE RESULTS IN ASSAYS FOR THE DETECTION OF ANTIBODIES AGAINST HIV

Reasons for False-Positive HIV Screening Test Results

• Increased sensitivity of assays, leading to reduced specificity • Technical errors • Presence of HIV antibodies in recipients of HIV-1 trial vaccines.Other rare possibilities: • Hypergammaglobulinemia/antibodies reactive to cellular components • Influenza vaccination may cause cross-reactivity with HIV antibody assays. The time course for such cross-reactivity remains uncertain.

Reasons for False-Negative HIV Screening Test Results

• Testing individuals during the window period (the incubation period between exposure and seroconversion)

• Technical errors • HIV-2 (for tests designed to detect only HIV-1). Other rare possibilities: • Delayed antibody synthesis in infants and persons

receiving post-exposure prophylaxis or with concurrent acute hepatitis C infection

• Diminished immune response in individuals receiving intensive or long-term immunosuppressive therapy

• Congenital or drug-induced hypogammaglobulinemia or agammaglobulinemia

• Insufficient host antibody response (i.e., advanced HIV disease)

• Unavailability of antibodies due to the formation of antigen-antibody complexes

• Reduced sensitivity assays

Reasons for Indeterminate* Western Blot Results

(positive screening and negative WB)

Probable True Positive (HIV Infection) • Seroconverting • HIV-2 infection • Technical errors Probable True Negative (No HIV Infection) • Recipients of HIV-1 trial vaccine • Antibodies reactive to cellular components, as in o Multiparous women o Polytransfused patients o Patients receiving chronic hemodialysis o Patients with autoimmune disease • Recipients of influenza and hepatitis B virus vaccines • Persons with non-HIV acute viral infections • Congenital bleeding disorders • Alcoholic hepatitis and other chronic liver diseases • Hematologic malignancies, lymphomas • Positive rapid plasma reagin test • Technical errors

Other lab tests for HIV patients

References: Centers for Disease Control and Prevention and Association of Public Health Laboratories.

Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. Available at http://stacks.cdc.gov/view/cdc/23447. Published June 27, 2014. Accessed [30/11/2015].

Diagnosis of HIV-1 Infection. Estelle Piwowar-Manning, HPTN Central Laboratory. The Johns Hopkins University.

Thank You