Human Immunodeficiency Virus - HIV
Laboratory 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 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
CD4 Count
200 - 500/mm3
<200/mm3
<100/mm3
<50/mm3
Infectious Complications
Bacterial pneumonia, Pulmonary
Tuberculosis, Herpes Zoster, Thrush,
Cryptosporidiosis, Kaposi’s sarcoma
Pneumocystis jiroveci (carinii),
Disseminated Histoplasmosis and
Coccidioidomycosis, Extrapulmonary TB
Disseminated Herpes Simplex Virus,
Toxoplosmosis, Cryptococcosis, Candida
esophagitis
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
– 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 Prophylaxis
Examples
Drug
Pneumocystis
jiroveci (PCP)
SXT
Start
Stop
CD4 < 200 CD4 >200
for 3-6 months
MAC
Azithromycin
CD4 < 50
M.
tuberculosis
Isoniazid
TST > 5mm
CD4 >100
for 3-6 months
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