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HIV:
Virology, Genomics &
Molecular Detection
Laboratorio de Biología Molecular
Facultad de Medicina UASLP
CA Garcia Sepúlveda MD PhD
1
Taxonomy
Virus classification is based on: phenotypic
characteristics, morphology, nucleic acid type, mode of
replication, host organisms, & the type of disease they
cause.
A combination of two main schemes is currently in
widespread use for the classification of viruses.
- Baltimore Classification
- International Committee on Taxonomy of Viruses
Baltimore Classification (David L. Baltimore), American biologist,
1975 Nobel Prize "for discoveries concerning the interaction between tumour
viruses and the genetic material of the cell."
Also NF-Kβ, and recombination activating genes RAG-1 and RAG-2.
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Taxonomy
Baltimore Classification Scheme
Based on the method used for viral
mRNA synthesis.
Seven groups depending on:
nucleic acid (DNA or RNA),
strandedness (ss or ds),
Sense (positive or negative), and
method of replication.
Those in a given category will all
behave in a similar fashion
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Taxonomy
Baltimore Classification Scheme
Group I: dsDNA viruses
Adenoviruses,
Herpesviruses,
Poxviruses, etc
Variola
HSV
Polyoma
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Taxonomy
Baltimore Classification Scheme
Group II: ssDNA viruses (+)sense
Parvoviruses
Parvo
5
Taxonomy
Baltimore Classification Scheme
Group III: dsRNA viruses
Reoviruses
Rotavirus
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Taxonomy
Baltimore Classification Scheme
Group IV: (+)ssRNA
Picornaviruses
Togaviruses
Poliovirus
Equine
Encephalitis
Rubella
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Taxonomy
Baltimore Classification Scheme
Group V: (-)ssRNA viruses
Orthomyxovirus
Rhabdovirus
Orthomyxo
Rhabdo
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Taxonomy
Baltimore Classification Scheme
Group VII: dsDNA-RT integrating
viruses
Hepadnavirus
HBV
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Taxonomy
Baltimore Classification Scheme
Group VI: (+)ssRNA-RT viruses with
DNA intermediate
Retrovirus
HIV-1
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Taxonomy
International Committee on Taxonomy of Viruses
Orders
Unassigned
Families Retroviridae
Subfamilies Orthoretrovirinae
Genera
Lentivirus
Species
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Taxonomy
International Committee on Taxonomy of Viruses
Family
Subfamily
Orthoretrovirinae
Retroviridae
Spumaretrovirinae
Genus
Alpharetrovirus
Betaretrovirus
Deltaretrovirus
Epsilonretrovirus
Gammaretrovirus
Lentivirus
Spumavirus
Species
Bovine leukemia virus
Primate T-lymphotropic virus 1
Primate T-lymphotropic virus 2
Primate T-lymphotropic virus 3
Bovine Immunodef virus
Caprine arthritis encephalitis virus
Equine infectious anemia virus
Feline immunodeficiency virus
Human immunodeficiency virus 1
Human immunodeficiency virus 2
Puma lentivirus
Simian immunodeficiency virus
Visna/maedi virus
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Taxonomy
International Committee on Taxonomy of Viruses
Family
Subfamily
Orthoretrovirinae
Retroviridae
Spumaretrovirinae
Genus
Alpharetrovirus
Betaretrovirus
Deltaretrovirus
Epsilonretrovirus
Gammaretrovirus
Lentivirus
Spumavirus
Species
Bovine leukemia virus
Primate T-lymphotropic virus 1
Primate T-lymphotropic virus 2
Primate T-lymphotropic virus 3
Bovine Immunodef virus
Caprine arthritis encephalitis virus
Equine infectious anemia virus
Feline immunodeficiency virus
Human immunodeficiency virus 1
Human immunodeficiency virus 2
Puma lentivirus
Simian immunodeficiency virus
Visna/maedi virus
13
Retrovirus
Key Features
A. Human viruses associated with tumors, leukemias &
immunodeficiencies.
B. Common genetic organization and strategy - major differences lie in
regulatory complexity.
C. Genome consists of 2 copies of (+) stranded RNA.
D. RNA converted to DNA by a bizarre mechanism followed by
chromosomal integration (reverse transcription) .
E. Integrated DNA co-linear with viral DNA.
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Central Dogma
Postulated by Francis Crick in 1958 and
restated in 1970.
3x3
3 Biopolymers: DNA, RNA y polipeptides.
3 Modalities general, special and unreported.
3 Directions for each modality.
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Central Dogma
3 General Directions:
DNA to DNA (Replication).
DNA to a RNA (Transcription).
RNA to AA (Translation).
3 Special Directions:
RNA to RNA (RNA Replication).
RNA to DNA (Reverse transcription).
DNA to AA (Direct translation, in vitro only).
3 Unreported Directions:
AA to DNA (Intelligent evolution).
AA to RNA (Intelligent evolution).
AA to AA (prions).
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Retrovirus
Examples
HTLV-1, HTLV-2… and HTLV-5 include members that can immortalize and
transform target cells.
- They are fast acting & cause sarcomas and leukemias through
protooncogenes (±35) which cause disregulation of cell growth.
- Hormones, growth hormone receptors, protein kinases, GTP-binding
proteins & nuclear DNA binding proteins.
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Retrovirus
Examples
HIV-1 and HIV-2 include members that are slow viruses and associated with
neurologic and immunosuppressive disease.
HIV is a slow cytocidal virus with exquisite tropism for CD4+ expressing cells
and macrophages.
It is the loss of CD4+ cells which destroys helper and delayed type
hypersensitivity functions of the immune response.
Human foamy virus (spumavirus) not associated with disease.
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Morphology
Primate lentiviruses have a distinct morphology and can induce syncytia
during productive infections.
Electron micrograph showing HIV-1 particles in the process of budding from
an infected cultured human PBMC, and several mature virions containing the
characteristic conical/bullet-shaped nucleoid.
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HIV
Lentiviral member of the retrovirus family.
Formerly:
Human T-lymphotropic virus-III (HTLV-III),
Lymphadenopathy-associated virus (LAV),
and AIDS-associated retrovirus (ARV).
Transmitted by blood, semen, vaginal fluid,
pre-ejaculate and/or breast milk.
Present in these body fluids both as free virus particles and as viral
particles within infected immune cells.
Four major routes of transmission are unprotected sexual intercourse,
contaminated needles, breast milk, and transmission from an infected
mother to her baby at birth.
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Morphology
HIV is different in structure from other
retroviruses (spherical)
Diameter of about 120 nm.
60 times smaller than a red blood cell,
but very large for a virus.
Contains two copies of positive singlestranded RNA that codes for the
virus's nine genes.
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Morphology
HIV is different in structure from other
retroviruses (spherical)
Diameter of about 120 nm.
60 times smaller than a red blood cell,
but very large for a virus.
Contains two copies of positive singlestranded RNA that codes for the
virus's nine genes.
Envelpoed by a matrix protein shell.
Protected by a conical capsid.
Stabilized by a nucleocapsid.
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Morphology
Organization of a mature human immunodeficiency virus (HIV)-1 virion.
Plasma membrane envelope
Surface envelope glycoprotein
Transmembrane envelope glycoprotein
Matrix protein (p17)
Capsid (p24)
Protease (p11)
Reverse Transcriptase (p66/p51)
Nucleocapsid (p7)
Viral RNA genome
Integrase (p31)
Vi
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Morphology
The single-stranded RNA is tightly bound to nucleocapsid proteins, p7
and enzymes needed for the development of the virion such as reverse
transcriptase, proteases, ribonuclease and integrase.
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Morphology
Envelope consists of a cap made of three molecules called glycoprotein
(gp) 120, and a stem consisting of three gp41 molecules that anchor the
structure into the viral envelope.
This complex enables the virus to attach to and fuse with target cells.
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Morphology
Functional relevance
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Morphology
Structural integrity of mature viral particle kept by matrix protein, capsid,
nucleocapsid and envelope.
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Morphology
Homing and attachment involve gp120 and gp41.
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Morphology
Fusion involves both matrix proteins (p17) and the envelope
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Morphology
Reverse transcription requires Reverse Transcriptase (p66/p51)
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Morphology
Integration requires intgrase (p31) and ribonuclease.
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Morphology
Expression and assembly requires protease (p11).
32
Genome
Genomic organization of simple and
complex retroviruses.
Simple retrovirus Moloney murine
leukemia virus (MuLV) contains:
Long terminal repeat (LTR) sequences
provide transcriptional regulatory
elements.
Gag encodes structural proteins of the
virus.
Pol encodes enzymes involved in
reverse transcription / integration.
Env encodes the virion surface
glycoproteins.
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Genome
Genomic organization of simple and
complex retroviruses.
Simple retrovirus Moloney murine
leukemia virus (MuLV) contains:
Long terminal repeat (LTR) sequences
provide transcriptional regulatory
elements.
Gag encodes structural proteins of the
virus.
Pol encodes enzymes involved in
reverse transcription / integration.
Env encodes the virion surface
glycoproteins.
Group Specific Antigen
Polymerase
Envelope
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Genome
Since HIV has a more complex life
cycle than simple retroviruses such as
MuLV one can expect a more complex
genome.
HIV can control its replication in a more
complex fashion.
HIV genome length is of 9749 bases
(average for retroviruses).
Where does the difference lie, where is
the rest of the complexity encoded?
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Genome
DUAL LAYER DVD !
Makes use of overlapping reading
frames (ORFs).
ORF: Open Reading Frame
1: ATG CAT GCA TGC ATG
2: TGC ATG CAT GCA TGC
3:
GCA TGC ATG CAT GCA
Same genome length, higher density!
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Genome
HIV-1 Encodes for more proteins than theoretically possible with a
single ORF.
Overlapping genes (such as
ENV, TAT and REV use
different ORFs of the SAME
genomic region.
Genes in different ORFs
(REV) can be split by other
genes (TAT).
HIV genome has nine open reading frames that code for 19 proteins.
Some of these EXTRA proteins are involved in transcriptional regulation.
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Genome
RNA genome consists of at least 7 structural landmarks (LTR, TAR,
RRE, PE, SLIP, CRS, INS) and nine genes (gag, pol, and env, tat, rev,
nef, vif, vpr, vpu, and tev) encoding 19 proteins.
38
Genome
Three of these genes, gag, pol, and env, contain information needed to
make the structural proteins for new virus particles.
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Genome
Gag region is 2000 bp and codes for the core structural proteins (matrix,
capsid & nucleocapsid).
Initial transcript size is p53, final products are p18, p24 and p15.
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Genome
Pol region is the longest at 2900 bp and codes for the enzymes.
Initial transcript size is p160.
Final cleavage products are p10 (protease), p66 (RT) and p32 (integrase).
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Genome
Env region is the shortest structural gene at 1800 bp and codes for the
surface and transmembrane glycoproteins.
Initial transcript size is p160.
Final cleavage products are gp120 and gp40.
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Genome
The six remaining genes, tat, rev, nef, vif, vpr, and vpu (or vpx in the
case of HIV-2), are regulatory genes for proteins that control the ability
of HIV to infect cells, produce new copies of virus (replicate), or cause
disease.
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Genome
The two Tat proteins (p16 and p14) are transcriptional transactivators
for the LTR promoter acting by binding the TAR RNA element
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Genome
The Rev protein (p19) is involved in shuttling RNAs from the nucleus
and the cytoplasm by binding to the RRE RNA element.
45
Genome
The Vif protein prevents the action of APOBEC3G (a cell protein which
deaminates DNA:RNA hybrids and interferes with the Pol protein).
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Genome
The Vpr protein (p14) arrests cell division at G2/M.
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Genome
The Nef protein (p27) downregulates CD4 (the major viral receptor), as
well as the MHC class I and class II molecules.
48
Genome
The Vpu protein (p16) influences the release of new virus particles
from infected cells.
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Reverse Transcription
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Reverse Transcription
A tRNA binds to PB site on the viral
RNA genome as its being transported
to cell nucleous.
Provides a free 3’ OH end for RT.
51
Reverse Transcription
5’ part of first DNA strand is
synthesized (blue) in the 3’  5’
direction (for reading), 5’  3’ direction
for writing.
Synthesis of DNA is followed by rapid
degradation of RNA (dotted line) due
to RT’s RNAse H function.
52
Reverse Transcription
DNA:tRNA hybrid is then transfered to
the 3’ end of the RNA genome.
R site:R site complementarity.
First strand synthesis proceeds.
RNA is further degraded EXCEPT the
PP site on viral RNA genome.
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Reverse Transcription
PP site serves as a primer for the
second strand synthesis (green).
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Reverse Transcription
PP site RNA and tRNA are degraded
and first and second strand PB sites
can hybridize to form a partially
circular preintegration genome.
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Reverse Transcription
PP site RNA and tRNA are degraded
and first and second strand PB sites
can hybridize to form a partially
circular preintegration genome.
Preintegration genome DNA is fully
duplex at end of process.
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Reverse Transcription
PP site RNA and tRNA are degraded
and first and second strand PB sites
can hybridize to form a partially
circular preintegration genome.
Preintegration genome DNA is fully
duplex at end of process.
Preintegration genome (and
postintegration genome also) is
colinear with viral RNA genome.
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Genetic Variability
HIV differs from many viruses in that it has
very high genetic variability.
This diversity is a result of its fast replication
cycle, with the generation of 109 to 1010
virions every day, coupled with a high
mutation rate of approximately 3 x 10-5 per
nucleotide base per cycle of replication and
recombinogenic properties of reverse
transcriptase.
This complex scenario leads to the
generation of many variants of HIV in a
single infected patient in the course of one
day!
58
Genetic Variability
This variability is compounded when a single
cell is simultaneously infected by two or
more different strains of HIV.
When simultaneous infection occurs, the
genome of progeny virions may be
composed of RNA strands from two different
strains.
This hybrid virion then infects a new cell
where it undergoes replication.
As this happens, the reverse transcriptase,
by jumping back and forth between the two
different RNA templates, will generate a
newly synthesized retroviral DNA sequence
that is a recombinant between the two
parental genomes.
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Genetic Variability
HIV-1 and HIV-2 are closely related.
HIV-2 is very closely relatd to SIVAGM
and SIVSm, SIVMac.
These SIV variants infect our closest
relatives Pan troglodytes troglodytes
(common) and Pan paniscus (bonobo).
Both HIV-1 and HIV-2/SIVPatr are
closely related to other SIV’s (gibbons,
maccaque, etc).
HIV’s and SIV’s are closely related to
other IV such as Feline
Immunodefficiency Virus (FIV).
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Genetic Variability
HIV-1 is divided into three viral clades
or subtypes based on genetical
differences of the env gene: M, N & O.
M is the most prevalent amongst
Hosa.
M subtype is further classified into
eight subtypes based on full-length
genomic differences (geographically
distinct).
circulating recombinant forms
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Genetic Variability
B, most prevalent in North and South America, Europe and Australia.
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Genetic Variability
CRFO2, AG recombinants, F, G, D, H, J, K and CRFO1 recombinants in
Africa.
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Genetic Variability
C, B, BC recombinants and CRFO1, AE, B recombinants in Asia.
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Genetic Variability
B,F Recombinants in South America.
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Genetic Variability
In 2006, the last year in which an analysis of global subtype prevalence
was made:
47.2 percent of infections worldwide were of subtype C,
26.7 percent were of subtype A/CRF02 AG,
12.3 percent were of subtype B,
5.3 percent were of subtype D,
3.2 percent were of CRF AE,
and the remaining 5.3 percent were composed of other subtypes and
CRFs.
Most HIV-1 research is focused on subtype B; few laboratories focus on
the other subtypes.[89]
66
HIV Testing Technologies
Use HIV rapid diagnostic test to identify an HIV infection
Initiate treatment with ARVs
minimal diagnostics required
Monitor effectiveness of ARVs with viral load, CD4
counts and safety with basic pharmacological laboratory
tests.
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HIV Testing Technology Spectrum
– HIV diagnosis (Antibody/Antigen testing)
• Enzyme Immunoassays (EIAs)
• Rapid tests
• Western blot (WB)
– Early diagnosis in infants
• P24
– Initiation and monitoring of ART
• CD4
• Viral Load
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HIV Testing Technology Challenges
– Early detection of seroconversion
– Early detection in infants born to HIV positive mothers
– Effect of HIV subtypes on test performance
– Impact of other health conditions on test performance
– Product specific equipment
– Technical skill
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Enzyme Immunoassays (EIAs)
– Quantitative assay to measure
HIV antibodies
• Most detect antibodies to
HIV-1 and HIV-2
• HIV Antigen / Antibody
reaction is detected by color
change
• Intensity of color reflects
amount of antibody present
serum
70
Enzyme Immunoassays (EIAs)
In an ELISA test, a person's serum is
diluted 400-fold and applied to a plate
to which HIV antigens have been
attached.
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Enzyme Immunoassays (EIAs)
If antibodies to HIV are present in the
serum, they may bind to these HIV
antigens.
The plate is then washed to remove all
other components of the serum.
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Enzyme Immunoassays (EIAs)
A specially prepared "secondary
antibody" — an antibody that binds to
human antibodies — is then applied to
the plate, followed by another wash.
This secondary antibody is chemically
linked in advance to an enzyme.
Thus the plate will contain enzyme in
proportion to the amount of secondary
antibody bound to the plate.
73
Enzyme Immunoassays (EIAs)
A substrate for the enzyme is applied,
and catalysis by the enzyme leads to a
change in color or fluorescence.
ELISA results are reported as a
number; the most controversial aspect
of this test is determining the "cut-off"
point between a positive and negative
result.
74
HIV Western Blot / Line Immunoassays
• Used as supplemental test for confirmation (only difficult cases)
• Detects antibodies to specific HIV antigens on cellulose strip
• Issues:
Multiple performance standards & interpretation
Expensive
Limited commercial availability
75
HIV p24 Antigen EIA
• Core protein of the virus
• EIA detects p24 antigen before antibody can be detected
 Detected 2 to 3 weeks after HIV infection
 Detected about 6 days before antibody tests become reactive
• Used for:
– Diagnosis of pediatric HIV-1 infections
– Blood bank safety (high incidence countries)
• Issues:
– Level 4 complexity (tehcnically demanding)
– Properly maintained equipment required
76
WHO Classification of HIV tests
Level 1: No additional equipment, little/no laboratory experience needed
Level 2: Reagent preparation or a multi-step process is required;
centrifugation or optimal equipment
Level 3: Specific skills such as diluting are required
Level 4: Equipment and trained laboratory technician are required
77
CD4 T-Lymphocyte Counts
• CD4 T-lymphocyte counts used for:
– Determining clinical prognosis
– Assessing criteria for
antiretroviral therapy
– Monitoring therapy
• Manual and automated methods
• Issues:
– Requires high level of technical
skill for test performance and
interpretation
– Properly maintained equipment
78
Viral Load
– Quantitative molecular assay measures amount of HIV in blood
products
– Used to:
• Predict disease progression
• Monitors response to anti-retrovirals
– Issues:
• Expensive
• Labor-intensive
• Special facilities
79
HIV Rapid Tests
– Qualitative assay to detect HIV antibodies
– Most detect HIV 1 and HIV 2 (do not distinguish them)
– As reliable as EIAs
– Issues:
• Small volumes
• Validation of use
• Appropriate training
80
HIV Rapid Tests Advantages
– Increases access to prevention/intervention
– Supports increased number of testing sites
– Same-day diagnosis and counseling
– Robust and easy to use
– Test time under 30 minutes
– Most require no refrigeration
– None or one reagent
– Minimal or no equipment required
– Minimum technical skill
81
HIV Rapid Tests Disadvantages
– Small numbers for each test run
– Quality Assurance/Quality Control at multiple sites
– Test performance varies by product
– Reader variability in interpretation of results
– Limited end-point stability of test results
82
HIV Rapid Tests Biospecimens
Biospecimens:
– Serum
– Plasma
– Whole blood
– Oral fluids
Formats:
– Immunoconcentration (flow-through device)
– Immunochromatography (lateral flow)
– Particle agglutination
83
HIV Rapid Tests Biospecimens
Formats:
– Immunoconcentration (flow-through device)
– Immunochromatography (lateral flow)
– Particle agglutination
Nonreactive
Reactiv
e
84
HIV Rapid Tests Biospecimens
Formats:
– Immunoconcentration (flow-through device)
– Immunochromatography (lateral flow)
– Particle agglutination
Add
Sample
IgG Antibodies
HIV antibodies
Conjugate
Test
Line
Colloidal gold
conjugated to HIV antigen
Control
line
Anti-IgG/gold
antibodies
HIV antigen
85
HIV Rapid Tests Biospecimens
Formats:
– Immunoconcentration (flow-through device)
– Immunochromatography (lateral flow)
– Particle agglutination
86
HIV Rapid Tests Biospecimens
Formats:
– Immunoconcentration (flow-through device)
– Immunochromatography (lateral flow)
– Particle agglutination
87
Window Period
The window period is the time from
infection until a test can detect any
change.
The average window period with
HIV-1 antibody tests is 22 days for
subtype B (3w to 6 m, avr 30d).
Antigen testing cuts the window
period to approximately 16 days
and NAT (Nucleic Acid Testing)
further reduces this period to 12
days.[2]
12 d
22 d
88
NAT (Nucleic Acid Testing)
Nonspecific reactions, hypergammaglobulinemia, or the presence
of antibodies directed to other infectious agents that may be
antigenically similar to HIV can produce false positive results with
serological testing.
Autoimmune diseases, such as systemic lupus erythematosus,
can also cause false positive results.
– Estrategia de mayor especificidad y sensibilidad teórica.
– Relativamente económica.
– Especialmente util para masificación de tamizajes.
– Brinda mucha más información que otro métodos (mayor resolución).
– Tiempo promedio de entrega de 1 o 2 días.
89
NAT (Nucleic Acid Testing)
A
90