Human Immunodeficiency Virus - HIV Laboratory Testing CLS 552 Application of Clinical Medical Microbiology & Immunology Karen Honeycutt, MEd, MLS(ASCP)CMSMCM

Human: infecting human beings What is HIV? Human: infecting human beings Immunodefficiency: decrease or weakness in the body’s ability to fight off infections Virus: a pathogen that only reproduces inside a living cell RNA, single stranded, enveloped virus Retrovirus: contains reverse transcriptase enzyme that converts RNA to DNA

Types of HIV HIV 1 Most common throughout the world HIV 2 Found in West Central Africa, parts of Europe and India Both produce the same patterns of illness HIV 2 disease progress slower than HIV 1

HIV Entry Into Cells Viral envelope protein (gp 120) binds to target cells with CD4 receptor CD4 T lymphocytes primary target cells Other cells with CD4 receptor: Macrophages Peripheral blood monocytes B lymphocytes (≈ 5%) HIV turns host cell into HIV replication factory

How is HIV Transmitted Unprotected sexual contact with infected partner Exposure of broken skin to infected blood or body fluids Transfusion with HIV infected blood products Tissue transplantation Injection with contaminated object Mother to child during pregnancy, birth or breastfeeding NOT by: saliva, respiratory droplets, insect vectors or close personal contact

Early Disease Progression ≈ 2-4 Weeks HIV localizes in lymphoid organs Viremia ensues after infection Rapid spread within first few weeks after infection ≈ 30 billion virus particles produced in first weeks of infection Acute retroviral syndrome: fever, fatigue, rash, headache, lymphadenopathy, pharyngitis, myalgias, nausea, vomiting, diarrhea, night sweats Resolves in a few days to a few weeks

HIV Antibody Development Detectable levels usually at 3 to 8 weeks after infection Time between infection and detectable antibody levels = ‘window period’ Serologic tests (looking for patient antibody) will be negative during window period Viremia greatly decreased due to antibody Patient usually asymptomatic Clinical latency (average 10 years) HIV continues to replicate in lymphoid tissue

Disease Progression Severity of illness determined by: Amount of virus in body Degree of immune suppression: CD4 lymphocyte counts decrease CD4 counts <500 usually become symptomatic, develop opportunistic infections

Acquired: come into possession of something new What is AIDS? Acquired: come into possession of something new Immune Deficiency: decrease or weakness in the body’s ability to fight off infections Syndrome: signs and symptoms occurring together characterizing a particular abnormality HIV infection leads to a weakened immune system. This makes a person with HIV vulnerable to a group of illness, e.g., opportunistic infections, that would not as easily affect a healthy person AIDS results when HIV infection progresses to an advanced stage, damaging the immune system to a point at which the body can no longer fight illness. AIDS is a syndrome because it is characterized by a group of illnesses Drugs are available which can treat HIV and AIDS. These drugs are called antiretrovirals (ARVs). They prevent the virus from replicating and slow the progress of the disease, but there is still no cure for AIDS or vaccine to prevent HIV transmission. AIDS is the final stage of the disease caused by infection with HIV.

Reasons for Testing for HIV Identify those with infection so antiviral therapy can be initiated Identify carriers who may transmit infection to others (blood & organ donors, pregnant women, sex partners) Monitor disease progression Evaluate treatment efficacy

Types of Testing Most common Less common Serology to detect patient antibody production to HIV components Nucleic acid testing (NAT) to detect HIV viral nucleic acid or characterize nucleic acid (resistance to antiviral drugs) Less common Detect HIV antigen (viral components) – usually used to screen blood products Culture: very difficult and dangerous to perform

CDC Recommendation: Opt-Out Testing Testing all persons aged 13 to 64 years in all health care settings Why? 250,000 in US unaware of HIV infection Informed consent: inform patient HIV testing will be part of routine testing Consent is inferred unless patient declines

Nebraska Law Requirements Still requires patient signature indicating patient is consenting to HIV testing prior to blood being drawn Bill in legislature (LB 462) to remove this restriction and follow CDC Opt-Out testing recommendations Research indicates more patients consent to testing if seen as a routine test instead of a test to target at-risk behavior

This is a summary of the revised recommendations for HIV Testing of Adults and Adolescents in Healthcare Settings. (slide)

(bullets from slide)

Testing Algorithm – Standard Serology Patient >2 years of age Perform screening test Enzyme immunoassay that will detect HIV antibodies in patient serum Sensitivity and specificity ≈ 99% Turn-around time = 1 to 2 days If positive, the EIA test is repeated in duplicate on the same specimen If 2 of 3 screen EIA tests are positive, confirmatory testing automatically performed

Testing Algorithm – Standard Serology Patient >2 years of age Confirmatory Testing – Western Blot Viral components are separated via electrophoresis on nitrocellulose strips Incubate patient serum with strips If antibody present, antigen-antibody complexes form on strip Strip is stained to visualize any antigen-antibody complexes Positive: if 2 of 3 specific antigen-antibody bands present Sensitivity & Specificity >99% Turn-around time varies: 1-7 days

Testing Algorithm – Standard Serology Patient >2 years of age Confirmatory Testing – Western Blot Example + = positive control (-) = negative control pt. = patient Patient WB = positive p24 and p120/160 bands present (Positive: if 2 of 3 specific antigen-antibody bands present) = Specific bands looked for

Testing Algorithm – Standard Serology Patient >2 years of age Patient is confirmed HIV positive if: 2 of 3 screening tests are positive with confirmatory test (Western Blot) also positive Test combination: >99% sensitive and >99.99% specific If screen + and confirmatory negative, then patient is not considered positive: Recommend follow up testing in 4 weeks Reasons for false positive and false negative HIV serology (see next two slides)

Examples That Can Cause False Positive HIV Serology Positive syphilis serology Some malignant blood and autoimmune disorders DNA viral infections Alcoholic hepatitis Chronic renal failure Renal transplantation

Examples That Can Cause False Negative HIV Serology Window period before seroconversion (most common) Immunosuppressive therapy Some malignancies Bone marrow transplantation Test systems that mainly detect antibodies to p24

HIV Point-of-Care Testing (POCT) Public health needs for rapid HIV Tests High rates of non-return for test results Need for immediate information or referral for treatment choices Perinatal settings Post-exposure treatment settings Screening in high-volume, high-prevalence settings

HIV Point-of-Care Testing (POCT) Rapid or POCT is performed at the time the patient is seen clinically Specimens: whole blood, saliva, urine Only FDA approved assays used in health care settings Results in 10-30 minutes Sensitivity and specificity ≈ 99% Considered a screen test If positive confirmatory testing recommended If negative usually no further testing recommended

HIV + Confirmed: Additional Testing Quantitative Plasma HIV RNA (Viral Load) Not FDA approved for confirmatory testing as 2-9% false positive rate Determine viral load ‘set point’ at time of diagnosis to monitor patient disease progression therapeutic response

HIV + Confirmed: Additional Testing CD4 Lymphocyte Count Adult normal range = 700 to 1100 cells/mm3 Results used to stage the disease Make therapeutic decisions When to start antiviral therapy When to start prophylaxis for specific opportunistic infections Indicator of prognosis

Correlation of Complications with CD4 Counts Infectious Complications 200 - 500/mm3 Bacterial pneumonia, Pulmonary Tuberculosis, Herpes Zoster, Thrush, Cryptosporidiosis, Kaposi’s sarcoma <200/mm3 Pneumocystis jiroveci (carinii), Disseminated Histoplasmosis and Coccidioidomycosis, Extrapulmonary TB <100/mm3 Disseminated Herpes Simplex Virus, Toxoplosmosis, Cryptococcosis, Candida esophagitis <50/mm3 Disseminated: Cytomegalovirus (CMV) Mycobacterium avium complex

Perinatal HIV Infection in Infants Utilize nucleic acid testing (NAT) Can’t utilize serology as mother’s IgG HIV antibody will cross the placenta Infant + if two HIV NATs positive at two different times Early antiviral therapy is recommended in HIV + infants

Antiretroviral Therapy (2008) HAART—highly active anti-retroviral therapy 23 approved antiretroviral agents Nucleoside Reverse Transcriptase Inhibitors Non-NRTIs Protease Inhibitors Entry & Fusion Inhibitors Integrase Inhibitors 5 fixed dose combinations Guidelines DHHS—Department of Health and Human Services IAS-USA—International AIDS Society - USA

Goals of HAART Clinical: prolong life and improve quality of life Virologic: undetectable viral load (<20-50 copies/mL) Immunologic: immune reconstitution (normal CD4 count) Therapeutic: combination of drugs (3 or 4) Epidemiologic: reduce HIV transmission

Starting Antiretroviral Therapy Start if: Patient symptomatic, an infant or pregnant HIV RNA >30,000 copies/ml CD4 count <350/mm3 Consider if: HIV RNA <5000 copies/ml, CD4 count 350-500 /mm3 HIV RNA 5000-30000 copies/ml, CD4 count >500 /mm3 Defer if: HIV RNA <5000 copies/ml, CD4 count >500 /mm3

Definition of Treatment Failure Virologic failure Viral load not below detectable levels (>50-400 c/mL) Side effects – patient not taking meds Immunologic failure CD4 count fails to increase 100 cells/mm3 per year Clinical failure >3 months post HAART and still having symptoms

HIV Resistance Testing Genotypic testing: HIV gene sequencing of the patient’s virus to detect mutations known to confer drug resistance Report out specific gene sequences with the drugs that the virus will be resistant to Reasons to perform When patient is first diagnosed as baseline At the start of HAART or switching drugs Determine if patient has been infected with other virus strains Treatment failures

Opportunistic Infections Pneumocystis jiroveci (carinii) - fungi Causes pneumonia (PCP) Detection via stains of BAL fluid, lung tissue Mycobacterium tuberculosis Lung and systemic disease Detection via culture Mycobacterium avium complex (MAC) Disseminated disease

Opportunistic Infections Cryptosporidium sp. - parasite Diarrhea Detection of organism is stool via microscopy or antigen detection Toxoplasma gondii - parasite Encephalitis, brain abscess Detection via serology (looking for antibody), staining tissue or NAT

Opportunistic Infections Candida sp. - yeast Thrush, vaginitis, esophagitis Detection with culture Cryptococcus neoformans - yeast Meningitis, pneumonia, disseminated disease Detection via culture or antigen detection in CSF Cytomegalovirus (CMV) Retinitis, pneumonia Detection via viral culture, NAT

Opportunistic Infections Prophylaxis Examples Drug Start Stop Pneumocystis jiroveci (PCP) SXT CD4 < 200 CD4 >200 for 3-6 months MAC Azithromycin CD4 < 50 CD4 >100 for 3-6 months M. tuberculosis Isoniazid TST > 5mm Opportunistic infections are never cured in HIV+ patients

Summary HIV: single-stranded, RNA, enveloped, retrovirus Infect CD4 positive cells: especially CD4 lymphocytes Serology: 2 of 3 screen tests positive followed by positive confirmatory test = HIV + Monitor: CD4 count, viral load, resistance testing CD4 count <500 = possible opportunistic infections <350 = probably initiate antiviral therapy