What is HIV?
CLS 511 Medical Microbiology
Human Immunodeficiency
d fi i
virus
i ‐ HIV
Laboratory Testing
• 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
Karen Honeycutt, M.Ed., MT(ASCP)SM
Types of HIV
• HIV 1
– Most common throughout the world
• HIV 2
– Found in West Central Africa,
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
h
• Peripheral blood monocytes
• B lymphocytes (≈ 5%)
• HIV turns host cell into HIV replication factory
How is HIV Transmitted
Early Disease Progression ≈ 2‐4 Weeks
• 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
• HIV localizes in lymphoid organs
• Viremia ensues after infection
• Rapid spread within first few weeks after infection
CLS 511 Medical Microbiology
Unit 21 HIV Testing
≈ 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
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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
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
– Clinical latency (average 10 years)
• HIV continues to replicate in lymphoid tissue
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,
donors pregnant
women, sex partners)
• Monitor disease progression
• Evaluate treatment efficacy
CLS 511 Medical Microbiology
Unit 21 HIV Testing
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
2
CDC Recommendation: Opt‐Out Testing
Nebraska Law Requirements
• Why? 250,000 in US unaware of HIV infection
• Informed consent: inform patient HIV testing
will be part of routine testing
• Consent
C
iis inferred
i f
d unless
l
patient
i
d
declines
li
• Still requires patient signature indicating
patient is consenting to HIV testing prior to
blood being drawn
• Bill in legislature 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
CLS 511 Medical Microbiology
Unit 21 HIV Testing
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
present, antigen‐antibody
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
3
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
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 + 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
– Monitor patient disease progression
– Monitor therapeutic response
CLS 511 Medical Microbiology
Unit 21 HIV Testing
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
>99.99%
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 Bakerman p. 311
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
CD4 Lymphocyte Count
• Results used to stage the disease
• Make therapeutic decisions
– When to start antiviral therapy
– When
Wh to start prophylaxis
h l i ffor specific
ifi
opportunistic infections
• Indicator of prognosis
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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
• See Bakerman p. 310
• Utilize nucleic acid testing (NAT)
• Can’t utlize 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
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
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
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
CLS 511 Medical Microbiology
Unit 21 HIV Testing
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Definition of Treatment Failure
• Genotypic testing: HIV gene sequencing of the
patient’s virus to detect mutations known to
confer drug resistance
• Virologic failure
– Viral load not below detectable levels
(>50‐400 c/mL)
– Report out specific gene sequences with the drugs
that the virus will be resistant to
• Side effects – patient not taking meds
• Immunologic failure
– CD4 count fails to increase 100
cells/mm3
HIV Resistance Testing
• Reasons to perform
per year
• Clinical failure
– >3 months post HAART and still having symptoms
– 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
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) or
staining tissue
Opportunistic Infections Prophylaxis
Examples
Drug
• 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
CLS 511 Medical Microbiology
Unit 21 HIV Testing
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
6
Summary
• HIV: single‐stranded, RNA, enveloped, retrovirus
• Infect CD4 positive cells: especially CD4
lymphocytes
• Serology: 2 of 3 screen tests positive followed by
positive confirmatoryy test = HIV +
p
• Monitor: CD4 count, viral load, resistance testing
• CD4 count
<500 = possible opportunistic infections
<350 = probably initiate antiviral therapy
CLS 511 Medical Microbiology
Unit 21 HIV Testing
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