Laboratory aspects of

Human Immunodeficiency
Virus (HIV) Infection

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 Family : Retroviridae
Subfamily : Lentivirus
• Lentivirus, which literally means slow
virus - it takes such a long time to
develop adverse effects in the body.
• Attacks the immune system.
• RNA virus, 120nm in diameter
• Envelope gp120 (present till the
death of the patient) & gp41
• Icosahedral symmetry
• Nucelocapsid
– Outer matrix protein (p17)
– Major capsid protein (p24)
– Nuclear protein (p7)

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Types of HIV

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HIV Life Cycle: Attachment Requires CD4
Receptor plus a Coreceptor

Dr.T.V.Rao MD 5
 Lifecycle of HIV

 HIV particles enter the body in a body fluid as it can

not survive without a support medium.
 The virus targets any cell expressing CD4, including T
helper cells, macrophages, dendritic cells and
monocytes.
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 Life Cycle of HIV
Reverse Transcription: Viral RNA
is converted into DNA by unique
enzyme reverse transcriptase.
Reverse transcriptase
RNA ---------------------> DNA
Reverse transcriptase is the
target of several HIV drugs: AZT,
ddI, and ddC.
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 Pathogenesis of HIV / AIDS
Infected T-Cell

HIV HIV Infected New HIV

T-Cell T-Cell Virus
Virus

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Effects of HIV on the immune system

3 areas:

1. Destruction of CD4+ T cells

population

2. Immune effects due to HIV infection

3. Progression of HIV infection to AIDS

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 Host’s immune responses
• Both humoral and cell-mediated immune responses
partially control the viral production but in this
process they destroy the infected CD4+T cells,
leading to a gradual decline of CD4+ T cells

• HIV-specific CTLs kill infected CD4+ T cells

• Apoptosis of infected cells

• Antibodies that recognize a variety of HIV antigens

are produced - Antibody dependent cell-mediated
cytotoxicity
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 Window Period

• This is the period of time after becoming

infected when an HIV test is negative

• 90 percent of cases test positive within

three months of exposure

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• Immune responses fail to erradicate all viruses.
• Viral load is maintained at low level
• Continuous decline of CD4+ T cells

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 High Risk Populations:
1. Homosexuals & bisexuals
2. IV drug users
3. Improperly screened transfusion
recipients
4. Sexual partners of persons infected with
HIV
5. Infants of HIV –infected mothers

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 Blood and Body fluids contain High
concentration of Viral particles

• Blood
• Semen/Vaginal
fluids (as high
as blood)
• Breast milk
• Pus from sores

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 Low concentrations of HIV
It is highly unlikely you will be
infected if you come into contact
with:
• Sweat
• Tears
• Urine
• Saliva (-highly possible if blood from
mouth sores is present) 16
 How is HIV not spread?
• Shaking hands
• Hugging
• Swimming pools
• Toilet seats
• Insect bites
• Donating blood

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 Resistance
• The virus are inactivated in 10 minutes at 600 C and in
seconds at 1000 C
• At room temperature survive for seven days
• HIV are inactivated in 10 minutes by treatment with :
– 50% ethanol
– 35% Isopropanol.
– 0.5% Lysol and paraformaldehyde
– 10% house hold bleach
– Hypochlorite solution at 0.5%

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 WHO clinical case definition for
AIDS in South-East Asia
• WHO clinical case definition for AIDS in South-East
Asia
Clinical AIDS in an adult is defined as an individual who
has been identified as meeting the two criteria A and B
below:
A. Positive test for HIV infection by two tests based on
preferably two different antigens.
B. Any one of the following criteria:
• - Weight loss of 10% body weight or cachexia, not known
to be due to a condition unrelated to HIV infection
- Chronic diarrhoea of one month's duration, intermittent
or constant

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 CDC Classification of HIV
• Category 1: > 500 cells/mm3 (or CD4% >
28%)
• Category 2: 200-499 cells/mm3 (or CD4%
14% -28%)
• Category 3: < 200 cells/mm3 (or CD4% <
14%)
• (CD4+ T-lymphocyte counts per microliter
of blood)

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 INDONESIA HIV TESTING POLICY
• Any HIV testing must be accompanied by a
pretest and posttest counseling services
(through VCT / PICT)
• Testing without consent – hindrance to the
control of the epidemic

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 Individuals Counseling
• Transmission
• Prevention
• Risk Factors
• Promotion of safe
behavior
• Voluntary & Confidential
• Clarifies test results
• Need for additional
testing
• Reportability of Positive
Test Results
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 Laboratory tests in HIV
management
Diagnostic :
(i)Screening tests - ELISA and simple/rapid tests.
(ii) Confirmatory tests -
Western Blot assay, Polymerase chain reaction
(PCR), Ligase chain reaction (LCR), Nucleic acid
based Sequence assays (NASBA) and some ELISA
tests.
Follow up :
CD4+ count / CD4+%
Others :
Routine tests, microbiological tests

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 Serologic Test of HIV
• Initial test for HIV is an indirect ELISA test /
rapid test
• Economic, rapid, performed easily, high
sensitivity and specificity
• Detects anti-HIV antibodies in patient serum
• Antibodies are generally detectable within 3
months of infection
• Antibodies are typically directed at the
envelope glycoproteins (gp120 and gp41)
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Absence of Antibodies to do not
confirm absence of HIV infection
• Absence of antibody, as in ‘window period’ does
not exclude the presence of the virus which can
be detected by PCR amplification approximately
ten days after infection
• Patient is highly infectious, despite testing HIV
antibody negative, HIV is replicating rapidly in all
body compartments.

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 Algorythm of HIV Testing
EIA/ELISA
2 Test
Negative Positive

No HIV Exposure HIV Exposure

Low Risk High Risk

Negative Repeat ELISA Run

Every 3 months Positive Confirmation
for 1 year Test

Repeat every
6 months for continued
Indeterminate Negative Positive
High risk behavior

Repeat at Repeat at
End Testing Negative 3 weeks 2-4 months HIV
28+
 Confirmatory tests of HIV
• Positive or indeterminate ELISA tests for anti-HIV
antibodies are confirmed by Western Blott

• PCR can also be used

• PCR detects pro-viral DNA or viral RNA
• It is highly sensitive and specific but is more costly than
ELISA
• Can be used to test infants born to HIV-infected mothers

• Or third serologic test using different method and/or

antigen
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Indirect ELISA test

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 Western Blotting
• Confirms HIV infection
• Proteins are separated by electrophoresis and
transferred to a nitrocellulose membrane
• The proteins are treated with antibodies (ab)
• A discrete protein band represents the specific
antigen
• The bands from a positive Western blot are from ab
binding to specific proteins and gp from the HIV
• The CDC recommends that the blot should be positive
for two of the gp24, gp41 and gp120/160 markers

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HIV Western blot

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 Rapid Tests
• ADVANTAGES:
• quicker to perform
– do not require specialized equipment or
trained personnel
– results delivered on the same day

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HIV Occupational Exposure
PREVENTION --- FIRST
• The health care provider and patient may
decide to use antiretroviral therapy after
weighing the risks and benefits
• Antiretroviral should not be used for those
with low-risk transmissions or exposures
occurring more than 72 hours after exposure

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 Prevention
• ABC
• Abstinence is the only
100 % effective
method of not
acquiring HIV/AIDS.
• Plus D :
– Donors pretest
– Drug user prevention

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 Monogamous relationship
• A mutually monogamous (only one sex partner)
relationship with a person who is not infected
with HIV
• HIV testing premarital is necessary to prove your
future husband / wife is not infected

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 Paediatric HIV Testing
Infants born to HIV infected mothers will
have antibodies to HIV in their serum as a
result of:
– maternal-fetal transfer during pregnancy
– breast-feeding
Positive serologic test can not differentiate
the presence of HIV or anti HIV  PCR

they may not necessarily be infected !

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 c t m e from
Pro te
A I D S 38