o 1.

Contains ribonucleic acid (RNA) as its nucleic acid

LABORATORY DIAGNOSIS OF HIV INFECTION o 2. Has a unique enzyme, called reverse transcriptase, which
transcribes the viral RNA into DNA.
HIV (HUMAN IMMUNODEFICIENCY VIRUS) ● Spherical particle (100 to 120 mm in diameter) contains an inner core
●Etiologic agent of AIDS (ACQUIRED IMMUNODEFICIENCY VIRUS) with two copies of single-stranded RNA surrounded by a protein coat
• HIV-1 was identified by the laboratories of Luc Montagnier or capsid and an outer envelope of glycoproteins embedded in a lipid
(1983) of France and Robert Gallo And Jay Levy (1984) of bilayer.
the United States.
STRUCTURAL GENES
● Four groups of isolates: ● HIV GENOME INCLUDES THREE MAIN STRUCTURAL GENES:
o Group M (the main or major group) - responsible for majority gag, env, and pol-and a number of regulatory genes
of HIV-1 infections worldwide ● HIV genes and their gene products:
o Nine subtypes or clades, designated a, b, c, d, f, g, h, j, and k. ● gag gene
o Group O (The Outlier Group) o Codes for p55, a precursor protein with a molecular weight of
o Group N 55 kd, from which four core structural proteins are formed: p6,
o Group P p9, p17, and p24
o HIV-2 (discovered in 1986) is less pathogenic and has a o Codes for nucleocapsid and core proteins
lower rate of transmission. ● env gene
● THREE ROUTES: o Codes for viral envelope proteins
1. Sexual transmission o Codes for glycoproteins gp160, gp120, and gp41
2. Transmission through blood o gp160 is precursor protein that is cleaved to form gp12- and
3. Maternal transmission gp41 (attachment and fusion on host cells)
o Intimate sexual contact - majority of cases: vaginal/anal o Characteristics of hi vp160 is precursor protein that is cleaved
intercourse to form
o Presence of other STDs increases the likelihood of o Gp120 forms the numerous knobs or spikes that protrude
transmission by disrupting, protective mucous membranes from the outer envelope
and activating immunologic cells in the genital areas o gp41 is a transmembrane glycoprotein that spans the inner
o Contact with blood or other body fluids - occurs mainly and outer membrane and attaches to Gp120.
through sharing of contaminated needles by intravenous drug ● pol gene
users • Codes for viral enzymes necessary for viral replication
o Increased risk of transmission: • Reverse transcriptase (p51)
▪ 1. Exposures involving a large quantity of blood, • Ribonuclease (RNAse H; p66) - an enzyme involved in the
hollow-bore needles placed directly into an artery or degradation of the original HIV RNA
vein, or deep tissue injury. • Integrase (p31) - an enzyme which mediates the integration
▪ 2. Source patient is in the acute or advanced stages of viral DNA into the genome of infected host cells
of HIV infection (amount of virus circulating in the • Protease (p10) - cleaves precursor proteins into smaller
bloodstream is high) active units used to make the mature virions
o Perinatally (from infected mother to infant) -
▪ 1. Can occur during pregnancy
▪ 2. By transfer of blood at the time of delivery
▪ 3. Through breastfeeding.
CHARACTERISTICS OF HIV
COMPOSITION OF THE VIRUS
● Genus Lentivirinae of the virus family Retroviridae (retrovirus)

CHARACTERISTICS OF HIV
● Homology between HIV-1 and HIV-2 is approximately 50%. (env
differs greatly)
VIRAL REPLICATION
1. Virus attaches to a susceptible host cell (host cell cd4 antigen and gp
120 glycoproteins on the outer envelope of virus)
T helper (Th) cells are the main target for HIV infection (express high
numbers of cd4 molecules on their surface and bind the virus with high
affinity)
T-tropic or X4 strains - HIV viruses that preferentially infect t cells; m-tropic
or r5 strains - strains that can infect both macrophages and t cells
2. Additional binding step thru co-receptors (chemokine receptors) that
promote fusion of the
HIV envelope with the plasma cell membrane.
● CXCR4 (required for HIV to enter t lymphocytes); ccr5 (required for
entry into macrophages)
3. Viral particle is taken into the cell and uncoating of the particle exposes the
viral genome.
4. Action of the enzyme reverse transcriptase produces complementary DNA
from the viral RNA.
5. Double-stranded DNA is synthesized and, with the help of the HIV
integrase enzyme, becomes integrated into the host cell's genome as a
provirus.
6. Viral DNA within the cell nucleus is then transcribed into genomic RNA and
messenger RNA (mRNA), which are transported to the cytoplasm.
(transcription)
7. Translation of mRNA occurs, with production of viral precursor proteins and
assembly of viral particles.
8. Intact virions bud out from the host cell membrane and acquire their
envelope during the process.
9. Precursor proteins are cleaved by the viral protease enzyme in the mature
virus particles. (viruses can proceed to infect additional host cells)
IMMUNOLOGIC MANIFESTATIONS
IMMUNE RESPONSES TO HIV
● Increased levels of p24 antigen and viral RNA in the host's
bloodstream - initially detected upon viral replication
● B Lymphocytes produce antibodies to HIV (detected in the host's
serum by 6 weeks after primary infection).
o 1. Antibodies directed against the gp41 transmembrane
glycoprotein
o 2. Antibodies to the gag proteins such as p24
o 3. Antibodies to the env, pol, and regulatory proteins
● Viral envelope proteins - most immunogenic (can induce production of
neutralizing antibodies)
● these prevent the virus from infecting neighboring cells.
● T-cell-mediated immunity (CD8+ cytotoxic T lymphocytes or
CYTOLYTIC T CELLS (CTLS)) appear within weeks of HIV infection -
associated with a decline in the amount of HIV in the blood during
acute infection
● Innate immune defenses - NK and dendritic cells
● EFFECT OF HIV ON THE IMMUNE SYSTEM
o Hindered by the rapid mutation
o Downregulating production of Class I MHC molec.
o Cells that can harbor HIV as a silent provirus
● Decrease in CD4 Th cell population is the hallmark feature of HIV
infection.
 ● Decreased effectiveness of both antibody- and cell-mediated immune
responses.
CLINICAL SYMPTOMS OF HIV INFECTION
ACUTE/EARLY STAGE OF INFECTION
● Rapid burst of viral replication (high levels of circulating virus/viremia
can be seen in the blood)
● HIV begins to disseminate to the lymphoid organs.
● Reduction in cd4 count, then returns to slightly decreased to normal

CLINICAL LATENCY
● Decrease in viremia
● Clinical symptoms are subtle or absent.
● Cd4 cell count remains stable, then progressively declines
● Long-term nonprogressors (LTNP) - have normal/mildly depressed
cd4 t-cell counts and low viral loads; asymptomatic for more than 10
years in the absence of art.

AIDS
● Final stage of infection
● Profound immunosuppression with very low numbers of cd4 t cells
● A resurgence of viremia
● Life-threatening infections and malignancies

HIV-infected individuals often demonstrate neurological symptoms

EARLY HIV INFECTION

● Forgetfulness poor concentration
● Apathy psychomotor retardation
● Withdrawal
LATE DISEASE
● Confusion
● Disorientation
● Seizures
● Dementia gait disturbances
● Ataxia paraparesis
● Stage 0 - with early HIV infection who had an initial confirmed
SYMPTOMS OF AIDS IN INFANTS INCLUDE: HIV-positive laboratory result followed by a negative or indeterminate
● Failure to thrive, persistent oral candidiasis, hepatosplenomegaly, HIV test result within a 6-month period.
lymphadenopathy, recurrent diarrhea, or recurrent bacterial infections o Can be reclassified in one of the other categories (1,2,3) 180
● Neurological findings may be present days or more after initial diagnosis
TREATMENT AND PREVENTION

● Administration of antiretroviral therapy (ART) to suppress viral
replication.
● More effective regimens involve a combination of drugs from at least
two of the antiretroviral drug classes "combination antiretroviral
therapy (CART) / highly active antiretroviral therapy (HAART)"
● Goal: to reduce the patient's HIV viral load to a level that is below the
detectable limit of quantitative plasma viral load assays
EFFECT OF CART
● A significant decline in the incidence of opportunistic infections, a
delay in progression to aids, and decreased mortality in patients who
have received treatment.
● Recommended for all HIV-infected persons at the time of diagnosis
● Has impact in reducing perinatal transmission
APPROACHES IN DEALING WITH HIV
1. Community-based education aimed at high-risk groups
2. Use of antiretroviral drugs for preexposure prophylaxis (prep) to
prevent transmission to individuals who are HIV-negative but at a high
risk of contracting the infection
3. PROPHYLACTIC THERAPY WITH ANTIRETROVIRAL DRUGS IS
ALSO OFFERED TO HEALTH-CARE WORKERS WHO MAY. HAVE
BEEN EXPOSED TO HIV THROUGH PERCUTANEOUS OR
MUCOUS MEMBRANE
VACCINE - ULTIMATE MEANS OF PREVENTING INFECTION
LABORATORY TESTING FOR HIV INFECTION
● SCREENING AND DIAGNOSIS
• HIV ANTIBODY TESTING is used in the initial diagnosis of
HIV infection.
• ELISA OR RAPID TEST KITS
• Western Blot Test - was the standard confirmatory test for
HIV; replaced.
• Serologic testing for p24 antigen and nucleic acid testing for
HIV RNA have been incorporated into the initial HIV testing.
● TESTING ALGORITHMS
• A combination of laboratory tests performed in sequence to
screen for the presence of HIV infection and resolve any
discrepant results
• 1989, ELISA for HIV-1 antibody, + sample being confirmed by
western blot/HIV-spec.
• Ifa.
• 2004, HIV-1/HIV-2 antibody test, + sample being confirmed by
western blot/IFA.
• 2014, combination IA (antibodies to hiv-1 and HIV-2 and
HIV-1 p24 antigen) - for adults and children older than 2 yrs
old
• If +, sample to undergo rapid/differentiation IA that
discriminates HIV-1 from hiv-2.
• If + on combination IA, (-) on 2nd test, sample is to undergo
NAT (nucleic acid testing).
1. It allows for earlier detection of infections, as the time between
exposure and detectable results on the hiv-1/2/p24 combo assay is
typically between 15 and 17 days.
2. It overcomes the limitations associated with use of the western blot
test. (a lengthy procedure that is typically performed only by
specialized reference laboratories)
3. Western blot testing is less sensitive than the initial ELISA used for
screening.
SEROLOGICAL TEST PRINCIPLES
ELISA AND CLIA
● ELISA - cornerstone of screening procedures for HIV bec:
o 1. Easy to perform
o 2. Can be adapted to test a large number of samples
o 3. Are highly sensitive and specific
Five generations of ELISA
1ST GENERATION
● Based on a solid-phase, indirect-assay system that detected
antibodies to only HIV-1
 ● HIV antibodies in patient serum were detected after binding to a solid
support coated with viral lysate antigens from HIV-1 cultured in
human t-cell lines, followed by addition of an enzyme-labeled
anti-human igg conjugate and substrate.
● Cons: prone to false-positive results caused by reactions with hla
antigens or other components from the cells used to culture the virus;
they were unable to detect antibodies to HIV-2.
2ND GENERATION
● Indirect binding assays that used highly purified recombinant or
synthetic antigens from both HIV-1 and HIV-2, rather than crude cell
lysates
● Cons: decreased sensitivity (samples containing antibodies to certain
subtypes of
● HIV that lacked the limited antigens used in the assays were tested)
3RD GENERATION
● Use the sandwich technique, based on the ability of antibody to bind
with more than one antigen.
● Available in ELISA and CLIA formats
● Antibodies in patient serum or plasma bind to recombinant HIV-1 and
HIV-2 proteins coated onto a solid phase. After washing, enzyme or
chemiluminescent-labeled HIV-1 and HIV-2 antigens are added and
bind to the already bound HIV-specific patient antibodies. Substrate
(or trigger solution, if CLIA is used) is added, and the color
development (or release of light with CLIA) is proportional to the
amount of antibody in the sample
● Improves sensitivity by simultaneously detecting HIV antibodies of
different immunoglobulin classes, including IgM
4TH GENERATION
● Can simultaneously detect HIV-1 antibodies, HIV-2 antibodies, and
p24 antigen
● Employ a sandwich ELISA or CLIA
● Patient serum is incubated with a solid phase onto which synthetic or
recombinant HIV-1 antigens, HIV-2 antigens, and a monoclonal
antibody to HIV-1 p24 have been attached.
● Following a wash step to remove excess sample, a conjugate
containing chemiluminescent- or enzyme-labeled anti-p24 and
HIV-1/HIV-2 antigens is added. After a second incubation and wash
step, the appropriate trigger solution or substrate/stop solution is
added and the relative light units released or optical absorbance is
measured.
● Used in the initial step of the 2014 laboratory testing algorithm
5TH GENERATION
● bead-based immunoassay that can detect both HIV antibodies and
antigens, in addition can differentiate between HIV-1 and HIV-2
infection.
Rapid tests for HIV antibodies
● Simple, rapid methods (provide results within 30 minutes); detect
antibodies to HIV-1 alone or to both HIV-1 and HIV-2; can be used on
serum, plasma, whole blood samples obtained by venipuncture or
fingerstick (for some kits, oral fluid
● Lateral flow or flow-through immunoassays that produce a
colorimetric reaction in the case of a positive result.
● Interpretation of the results is made through visual observation of the
test device and does not require instrumentation
● Highly sensitive and specific
● Cons: false positives and false negatives can occur
● Western blot test (1984)
o Most common method used for systematic confirmation of
positive ELISA results from 1985 to 2014
o 1. Nitrocellulose or nylon strips containing individual HIV
proteins blotted onto the test membrane
o 2. Any HIV antibodies present in the sample will bind to their
corresponding antigens on the test membrane.
o 3. Washing procedure. (unbound ab is removed)
o 4. Anti-human immunoglobulin with an enzyme label is added
directly to the test strip and binds to specific HIV antibodies
from the patient sample.
o 5. Washing procedure. (unbound conjugate is removed)
o 6. Bound conjugate is detected after adding the appropriate
substrate. (producing chromogenic reaction)
● Antibodies to the gag proteins p24 and p55
o Appear early after exposure to the virus, but decrease or
become undetectable as clinical symptoms of aids appear.
● Antibodies to the envelope proteins gp41, gp120, and gp160
o Appear slightly later but remain throughout all disease stages
in an HIV-infected individual
● A more reliable indicator of the presence of HIV
Visual interpretation and densitometry
● Visual interpretation and densitometry can be performed to identify
the number and types of antibody produced.
● Performed only in specialized reference laboratories.
● Positive and negative control sera must be included in the test run
(quality control)
o Valid if:
 ▪ The negative control should produce no bands
▪ The positive control should be reactive with p17, p24,
p31, gp41, p51, p55, p66, and gp120/160
● Negative test result for the patient sample is reported if either no
bands are present or if none of the bands present correspond to the
molecular weights of any of the known viral proteins.
● Specimens that have some of the characteristic bands present but do
not meet the criteria for a positive test result are considered to be
indeterminate. (false positive caused by cross-reacting antibodies)
● Antibodies to the gag proteins p24 and p55
o Appear early after exposure to the virus, but decrease or
become undetectable as clinical symptoms of aids appear.
● Antibodies to the envelope proteins gp41, gp120, and gp160
o Appear slightly later but remain throughout all disease stages
in an HIV-infected individual
CONS OF WESTERN BLOT:
● Relative insensitivity
● level of technical difficulty
● long turnaround time to obtain results
CDC has recommended that the western blot be replaced with rapid
HIV-1/HIV-2 antibody tests as the standard method for confirmation of positive
screening results.
QUALITATIVE NUCLEIC ACID TESTS (NAT)
● Used to determine whether or not a detectable level of HIV nucleic
acid is present in human plasma.
● Used to screen for infection or make an initial patient diagnosis
● If serological results are inconclusive such as in very early infection or
in the diagnosis of infants, where presence of maternal antibodies can
confuse test results
● Used to resolve discrepancies between a positive result in the initial
antigen-antibody combo assay and the follow-up HIV-1/HIV-2
antibody differentiation assay
● HIV-1 antibody/HIV-2 antibody/p24 antigen test is nonreactive, but the
NAT is reactive, then this result is considered evidence for acute
HIV-1 infection.
● HIV-1/HIV-2 antibody test is indeterminate and the NAT is reactive,
this suggests that HIV-1 antibodies were indeed present
● HIV-1/HIV-2 antibody test is reactive or indeterminate and the NAT is
nonreactive, the initial test is a false-positive one.
● Qualitative Polymerase Chain Reaction (PCR)-based assay to screen
donors of whole blood, blood
QUANTITATIVE VIRAL-LOAD ASSAYS
● measure the amount of circulating HIV nucleic acid
● play an essential role in helping physicians
○ 1. predict disease progression
○ 2. monitor patient response to antiretroviral therapy, and
○ 3. guide treatment decisions
● HIV RNA levels become detectable about 11 days after infection and
rise to very high levels during the initial burst of viral replication.
● In period of a few months, the viral load drops as the individual's
immune system clears viral particles from the circulation and a stable
level of plasma HIV RNA ("set point") is achieved.
● Untreated indiv. - level can persist for a long time and rise again later
as the immune system deteriorates and the patient progresses to
AIDS.
● Successful therapy with ART - drop in the viral load to an
undetectable level, usually by 8 to 24 weeks after initiation of ART.
DISEASE MONITORING
CD4 T-CELL ENUMERATION
● CD4 lymphopenia - decrease because of destruction of CD4
T-lymphocytes.
HALLMARK FEATURE OF AIDS
● Normal count: 450 to 1,500 cells/uL (average 1,000 cells/uL)
CLINICAL APPLICATION:
1. CDC classification system uses CD4 T-cell counts to place patients
into various stages of HIV infection.
According to US Department of Health and Human services
● plasma HIV RNA testing be performed before antiretroviral therapy
begins to obtain a baseline value; testing should be performed
periodically thereafter to determine the effectiveness of the therapy.
METHODS - based on amplification methods that increase the number of HIV
RNA copies (or their derivatives) in test samples to detectable levels.
1. PCR
2. branched chain DNA assay (bDNA)
3. nucleic acid sequence-based amplification (NASBA) - amplifies HIV
RNA; not suitable for clinical laboratory settings
CD4 T-CELL ENUMERATION
 ● FLOW CYTOMETRY - gold standard for enumerating CD4 T cells
● Ratio of CD4 T cells to CD8 T cells may be reported to assess the
dynamics between the two T-cell populations.
● Has led to the manufacture of miniaturized flow cytometers that can
be easily operated by smaller laboratories.
● Disposable test cassettes are used to capture CD4+ cells with labeled
antibodies, and the results are analyzed by small, portable
instruments that can be powered by a rechargeable battery.
2. To monitor the effectiveness of antiretroviral therapy.
3. To help determine whether a change in ART is necessary and if
prophylactic drugs for certain opportunistic infections should be administered
● Annual measurements can be performed in patients whose CD T-cell
counts have consistently remained between 300 and 500 cells/j.L.
● Decline in the CD4 T-cell count of greater than 25%, the physician
may change the CART.
● Patients with a CD4 T-cell count below 200/uL are placed on
prophylactic therapy for Pneumocystis jiroveci pneumonia.
● Patients with a count of less than 50/pL are given prophylactic
treatment for Mycobacterium avium complex.
1. PCR RT-PCR
● amplify a DNA sequence that is complementary to a portion
of the HIV RNA genome
● limited dynamic range; highly susceptible to
cross-contamination with extraneous nucleic acid
2. quantitative (real-time) PCR (gPCR)
● detect and quantify the PCR products as they are being
produced
● highly sensitive and can detect a broad range of RNA copies
3. Branched-chain DNA assay
● based on amplifying the detection signal generated in the
reaction
● higher reproducibility and higher throughput
ALTERNATIVES TO CONVENTIONAL VIRAL LOAD TESTS
● point-of-care devices such as closed cartridges that provide
semiquantitative analysis with simpler instrumentation in a shorter
period of time.
○ loop-mediated and helicase-dependent amplification

DISEASE MONITORING
● signs of disease progression can be detected early and guide
decisions about further treatments.

TWO LABORATORY MARKERS:

● (1) peripheral blood CD4 T-cell count - best indicator of immune
function in HIV-infected individuals
● (2) HIV-1 RNA level/"viral load" - reflects patient responses to ART

TESTING OF INFANTS YOUNGER THAN 18 MONTHS

● A child born to an HIV-positive mother may test positive for HIV

antibody during the first 18 months of life even though the child is not
infected.
● HIV infection in infants is best diagnosed using molecular methods.
● qualitative HIV-1 DNA PCR test (detects proviral DNA within the
infants' peripheral blood mononuclear cells) - preferred method
● quantitative HIV RNA assays (less sensitive than DNA assays, RNA
tests can be used to provide a baseline viral load measurement) - can
be used as a confirmatory test for infants who initially had a positive
HIV DNA test.

METHODS