Human Immunodeficiency Virus - HIVLaboratory Testing

CLS 552 Application of Clinical Medical Microbiology & Immunology

Karen Honeycutt, MEd, MLS(ASCP)CMSMCM

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

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

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

– 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

Testing Algorithm – Standard SerologyPatient >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 SerologyPatient >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 SerologyPatient >2 years of age

• Confirmatory Testing – Western Blot Example

+ = positive control(-) = negative controlpt. = 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 SerologyPatient >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 TestingQuantitative 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 TestingCD4 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 CountsCD4 Count 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 pregnantHIV RNA >30,000 copies/mlCD4 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– Detection via culture

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 ProphylaxisExamples

Drug Start StopPneumocystis 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