13. Retroviridae

7/30/2019 13. Retroviridae

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Retroviridae

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Retroviruses

Use reverse transcription of viral RNA into DNA during

replication

Members of this family include HIV, feline leukemia, and

several cancer-causing viruses

Retroviruses were discovered in 1908 by Vilhelm Ellermann

and Oluf Bang.

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Genome

The genome is unsegmented, contains a single molecule of

linear structure.

Contain unique enzyme reverse transcriptase - RNA

dependent DNA polymerase, produces a dsDNA copy of the

viral RNA.

Positive-sense (2 copies of a +ve sense RNA)

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Genome

ssRNA approximately 10 kilo bases long.

The complete genome of one monomer is 7000 -

11000 nucleotides long

Retrovirus genomes commonly contain 3 open

reading frames that encode for group proteins that

can be found in the mature virus:

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Genome

gag (group-specific antigen)- codes for coreand structural proteins of the virus; They aretype specific & they define the individualvirus species.

pol (polymerase)- codes for reversetranscriptase, protease and integrase;

env (envelope)- codes for the retroviral coat

proteins. The gene order in all retroviruses is: 5-gag -

pol - env - 3

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http://en.wikipedia.org/wiki/Group-specific_antigenhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Group-specific_antigenhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Polymerasehttp://en.wikipedia.org/wiki/Reverse_transcriptasehttp://en.wikipedia.org/wiki/Polymerasehttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Integrasehttp://en.wikipedia.org/wiki/Reverse_transcriptasehttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Integrasehttp://en.wikipedia.org/wiki/Viral_envelopehttp://en.wikipedia.org/wiki/Viral_envelopehttp://en.wikipedia.org/wiki/Viral_envelopehttp://en.wikipedia.org/wiki/Viral_envelopehttp://en.wikipedia.org/wiki/Viral_envelopehttp://en.wikipedia.org/wiki/Integrasehttp://en.wikipedia.org/wiki/Proteasehttp://en.wikipedia.org/wiki/Reverse_transcriptasehttp://en.wikipedia.org/wiki/Reverse_transcriptasehttp://en.wikipedia.org/wiki/Polymerasehttp://en.wikipedia.org/wiki/Polymerasehttp://en.wikipedia.org/wiki/Polymerasehttp://en.wikipedia.org/wiki/Polymerasehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Group-specific_antigenhttp://en.wikipedia.org/wiki/Group-specific_antigenhttp://en.wikipedia.org/wiki/Group-specific_antigen


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Virion structure

The virions of a retroviridae consist of an envelope, a

nucleocapsid and a nucleoid

They replicate via a DNA intermediate.

Retroviruses rely on the enzyme reverse transcriptase to

perform the reverse transcription of its genome from RNA

into DNA, which can then be integrated into the host's

genome with an integrase enzyme.

The virus then replicates as part of the cell's DNA

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Provirus

The DNA can be incorporated into host genome as a

provirus that can be passed on to progeny cells.

In this way some retroviruses can convert normal

cells into cancer cells.

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Replication

Retrovirus virions enter host cells through interaction

between a virally-encoded envelope protein and a cellular

receptor.

Viral RNA is transcribed into a DNA copy by the enzyme RT

which is present in the virion.

The viral DNA copy is integrated into and becomes a

permanent part of the host genome.

This integrated DNA is provirus.

The host cell's transcriptional & translational machinery expresses theviral genes

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Replication

The host RNA polymerase II transcribes the provirus to create

new viral RNA, which is then transported out of the nucleus

by other cellular processes.

A fraction of these new RNAs are spliced to allow expression

of some genes, while others are left as full-length RNAs.

Viral proteins are synthesized by the host cell's translational

machinery.

Virions are assembled and bud from the host cell.

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Classification

Retroviruses are RNA viruses that contain the RT enzyme &

replicate in a unique manner.

They cause tumors in several species of animals.

Human retroviruses are found in 2 families: Oncovirinae = RNA tumour viruses which include

Human T cell lymphotropic virus HTLV-I & II

Lentivirinae = which cause chronic infections & these

include: HIV- It differs from oncovirinae in being cytolytic

(cytocidal) & non transforming (non-oncogenic).

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Human T-Cell Lymphotrophic Viruses, Types

1 and 2

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HTLV-I & II

HTLV-1 and -2 share about 65 percent nucleotide sequence

homology, and therefore are genetically and biologically

similar.

However, their worldwide distribution is different.

HTLV-1 has been associated with adult T-cell leukemia (ATL)

& HTLV-associated myelopathy (HAM).

HTLV-2 infection causes a rare form of cancer, hairy cell

leukemia

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Transmission of HTLV

The distribution of HTLV infection varies greatly with geographic

area and socioeconomic group.

For example, the overall incidence of antibody in United Statesblood donors is about 1 in 2500, whereas about 25 percent of New

York City intravenous drug users are infected with either HTLV-1 or

-2.

HTLV-2 infection is also high among the Native American population

of the southwestern United States.

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Transmission of HTLV

HTLV transmission occurs primarily by cell-associated virus, via one of

three routes.

1) in highly endemic regions, mother to fetus or newborn is the most

common mode of transmission.

2) infection can be transmitted sexually by infected lymphocytes contained

in semen.

3) any blood products containing intact cells are also a potential source of

infection.

There is little evidence for transmission by cell-free fluids 14


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Pathogenesis of adult T-cell leukemia

HTLV-1 infection both stimulates mitosis and immortalizes T lymphocytes,

which acquire an antigen-activated phenotype.

Following infection, the virus becomes integrated in the host cell as a

provirus and transforms a polyclonal population of T cells.

Although these cells all have an integrated provirus, there is no common

integration site in different tumors.

No HTLV mRNA is transcribed and no recognized oncogene is activated.

However, in the course of continued multiplication over a period of many

years, the infected T cells accumulate .

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Clinical significance of adult T-cell leukemia

The majority of infected individuals are asymptomatic carriers who have

an estimated one percent chance of developing adult T-cell leukemia (ATL)

within their lifetime.

ATL typically appears twenty to thirty years after initial infection, when an

increasingly larger population of monoclonal malignant ATL cells develops

and infiltration of various visceral organs by these cells occurs.

There is accompanying impairment of the immune system leads to

opportunistic infections

[ cytomegalovirus , P. jiroveci , and/or various disseminated fungal and16


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Pathogenesis and clinical significance of HTLV-

associated myelopathy

HTLV-associated myelopathy (HAM) is distinctly different from

ATL in that it usually appears only a few years after infection.

CNS involvement is indicated by:

1) the presence of anti-HTLV-1 antibody in the CSF;

2) lymphocytic infiltration and demyelination of the thoracic spinal

cord; and

3) brain lesions.

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Pathogenesis and clinical significance of HTLV-

associated myelopathy

HAM occurs with lower frequency than ATL among HTLV-

infected populations.

It is characterized by progressive spasticity and weakness ofthe extremities, urinary and fecal incontinence, hyperreflexia,

and some peripheral sensory loss.

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Pathogenesis and clinical significance of hairy

cell leukemia

This disease, caused by HTLV-2, is a rare, lymphocytic

leukemia that is usually ofB cell origin.

It is characterized by malignant cells that look ciliated

These cells replace bone marrow and infiltrate the spleen,

causing splenomegaly

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Laboratory identification

Screening of blood donors for HTLV is done by ELISA or

agglutination tests , but the existence of false-positives

necessitates confirmatory testing by Western blotting.

Test sensitivity is also a problem caused by the low and

variable antibody titers in infected individuals

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Treatment and prevention

The usual agents used in cancer chemotherapy have proven

to be ineffective in treating ATL, and attempts to treat HAM

have for the most part been equally unsuccessful.

Screening of blood donors and safe sex can effectively

prevent transmission .

No vaccine is currently available for human use,

But trials of experimental vaccines are in progress

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Human Immunodeficiency Virus

(HIV)

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SUB-SAHARAN AFRICA: 67.1%24


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HIV prevalence in Ethiopia

National HIV prevalence: 2.4% for 2010 Female: 2.9% Male: 1.9%

Urban HIV prevalence : 7.7%

Rural HIV prevalence : 0.9%

The highest HIV prevalence in Ethiopia occurs in the

age group 15-24

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The HIV Epidemic Unfolds

Sudden outbreak in USA of opportunistic infections andcancers in homosexual men in 1981

Pneumocystis carinii pneumonia (PCP), Kaposis sarcoma,and non-Hodkins lymphoma

HIV isolated in 1984 - Luc Montanier (Pasteur Institute, Paris)and Robert Gallo (NIH, Bethesda, USA)

HIV diagnostic tests developed in 1985

First antiretroviral drug, zidovudine, developed in 1986

Exploding pandemic Has infected more than 50 million people around the

world

Has killed over 22 million people

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Classification of HIV

HIV class: Lentivirus

Retrovirus: single stranded RNA transcribed to double stranded DNAby reverse transcriptase

Integrates into host genome

High potential for genetic diversity

Can lie dormant within a cell for many years, especially in resting(memory) CD4+ T4 lymphocytes

HIV type (distinguished genetically)

HIV-1 -> worldwide pandemic (current ~ 33.2 M people) HIV-2 -> isolated in West Africa; causes AIDS much more slowly than

HIV-1 but otherwise clinically similar

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HIV-1 and HIV-2 Differ in Multiple Ways

Accessory genes

HIV-1 vpu

HIV-2 vpx

Distribution

HIV-1 global pandemic

HIV-2 West Africa

Rate of progression of severe immunosuppression

HIV-1 median time to AIDS = 10 years

HIV-2 median time to AIDS = longer

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How many subtypes of HIV-1 are there?

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Classification of HIV-1

HIV-1 groups

M (major): cause of current worldwide epidemic

O (outlier) and N (Cameroon): rare HIV-1 groups that aroseseparately

HIV-1 M subgroups (clades)

>10 identified (named with letters A to K)

Descended from common HIV ancestor

One clade tends to dominate in a geographic region

Clades differ from each other genetically

Different clades have different clinical and biologicbehavior

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HIV-1 rapidly evolves by two mechanisms:

Mutation: changes in single nucleosides of the RNA

Recombination: combinations of RNA sequences from twodistinct HIV strains

Several common clades (e.g., A/G ad A/E) arerecombinants

Geographic distribution of HIV group M clades

A in Central Africa B in North American, Australia, and Europe

C in Southern and Eastern Africa (Ethiopia)

Origin and Distribution of HIV-1 Clades

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gp120 env protein binds to CD4 molecule

CD4 found on T-cells , macrophages, and microglial cells

Binding to CD4 is not sufficient for entry

V3 loop of gp120 env protein binds to co-receptor

CCR5 receptor - used by macrophage-tropic HIV variants

CXCR4 receptor - used by lymphocyte-tropic HIV variants

Binding of virus to cell surface results in fusion of viral envelope with cell

membrane

Viral core is released into cell cytoplasm

How HIV Enters Cells

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Viral-Host Dynamics

About 1010 (10 billion) virions are produced daily

Average life-span of an HIV virion in plasma is ~6 hours

Average life-span of an HIV-infected CD4 lymphocytes is ~1.6

days

HIV can lie dormant within a cell for many years, especially in

resting (memory) CD4 cells, unlike other retroviruses

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HIV Immunology

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Host: mounts HIV-specific immune responses

Cellular (cell-mediated) - most important

Humoral (antibody-mediated)

Virus: subverts the immune system

Infects CD4 cells that control normal immune responses

Integrates into host DNA

High rate of mutation Hides in tissue not readily accessible to immune system

General Principles of

Viral-host Interactions:

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CD8 Cytotoxic T lymphocyte (CTL)

Critical for containment of HIV

Derived from nave T8 cells, which recognize viral antigens

in context of MHC class I presentation

Directly destroy infected cell

Activity augmented by Th1 response

Cellular Immune Responses to HIV

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Cellular Immune Responses to HIV

CD4 Helper T Lymphocyte (Th)

Plays an important role in cell-mediated response

Recognizes viral antigens by an antigen presenting cell

(APC)

Utilizes major histocompatibility complex (MHC) class II

Differentiated according to the type of help

Th1 - activate Tc (CD8) lymphocytes, promoting cell-

mediated immunity Th2 - activate B lymphocytes, promoting antibody

mediated immunity

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Neutralization

Antibodies bind to surface of virus to prevent

attachment to target cell

Antibody-dependent cell-mediated cytotoxicity(ADCC)

Fc portion of antibody binds to NK cell

Stimulates NK cell to destroy infected cell

Humoral Immune Response to HIV

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HIV Evasion Methods

Makes (1010 ) 10 billion copies/day -> rapid mutation of HIV

antigens

Integrates into host DNA

Depletes CD4 lymphocytes

Down-regulation of MHC-I process

Impairs Th1 response of CD4 helper T lymphocyte

Infects cells in regions of the body where antibodies

penetrate poorly, e.g., the central nervous system

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Pathogenesis of HIV

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Numerous organ systems are infected by HIV:

Brain: macrophages and glial cells

Lymph nodes and thymus: lymphocytes and dendritic cells

Blood, semen, vaginal fluids: macrophages Bone marrow: lymphocytes

Skin: langerhans cells

Lung: alveolar macriphages

Cells Infected by HIV

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General Mechanisms of HIV Pathogenesis

Direct injury

Nervous (encephalopathy and peripheral

neuropathy)

Kidney (HIVAN = HIV-associated nephropathy)

Cardiac (HIV cardiomyopathy)

Endocrine (hypogonadism in both sexes)

GI tract (dysmotility and malabsorption)

Indirect injury

Opportunistic infections and tumors as a

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General Principles of

Immune Dysfunction in HIV

All elements of immune system are affected

Advanced stages of HIV are associated with substantial

disruption of lymphoid tissue

Impaired ability to mount immune response to new

antigen

Impaired ability to maintain memory responses

Loss of containment of HIV replication

Susceptibility to opportunistic infections

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Direct

Elimination of HIV-infected cells by virus-specific

immune responses

Loss of plasma membrane integrity because ofviral budding

Interference with cellular RNA processing

Indirect Syncytium formation

Apoptosis

Autoimmunity

Mechanisms of CD4

Depletion and Dysfunction

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Observed in HIV infection, most commonly in the brain

Uninfected cells may then bind to infected cells due to viral gp

120

This results in fusion of the cell membranes and subsequent

syncytium formation.

These syncytium are highly unstable, and die quickly.

Syncytium Formation

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Apoptosis

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Consequence of Cell-mediated


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Consequence of Cell-mediated

Immune Dysfunction

Inability to respond to intracellular infections and

malignancy

Mycobacteria, Salmonella, Legionella

Leishmania, Toxoplama, Cryptosporidium,Microsporidium

PCP, Histoplamosis

HSV, VZV, JC virus, pox viruses EBV-related lymphomas

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Natural History of

HIV Infection

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Primary HIV Infection

The period immediately after infection characterized by high levelof viremia (>1 million) for a duration of a few weeks

Associated with a transient fall in CD4

Nearly half of patients experience some mononucleosis-likesymptoms (fever, rash, swollen lymph glands)

Primary infection resolves as body mounts HIV-specific adaptive

immune response Cell-mediated response (CTL) followed by humoral

Then patient enters clinical latency

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Natural History of HIV-1

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Transmission

Modes of infection

Sexual transmission at genital or colonic mucosa

Blood transfusion

Mother to infant Accidental occupational exposure

Viral tropism

Transmitted viruses is usually macrophage-tropic Typically utilizes the chemokine receptor CCR5 to

gain cell entry

Patients homozygous for the CCR5 mutation are

relatively resistant to transmission 54


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MTCT of HIV

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Developing countries 40%

On Zidovudine alone 7%

Zidovudine with C-section 2%

HAART


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Viral load

Marker of HIV replication rate

Number of HIV RNA copies/mm3 plasma

CD4 count

Marker of immunologic damage

Number of CD4 T-lymphocytes cells/mm3 plasma

Median CD4 count in HIV negative Ethiopians issignificantly lower than that seen in Dutch controls

Female 762 cells/mm3

Male 684 cells/mm3

Laboratory Markers of HIV Infection

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HIV RNA Set Point Predicts


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HIV RNA Set Point Predicts

Progression to AIDS

HIV RNA viral loads after infection can be used in the

following ways:

To assess the viral set point

To predict the likelihood of progression to AIDS inthe next 5 years

The higher the viral set point:

The more rapid the CD4 count fall The more rapid the disease progression to AIDS

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P tt f HIV Di P i


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Patterns of HIV Disease ProgressionTypical

Progressors

7-10 years

HIVInfection Rapid Progressors 10-20 yr

85-90 %


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Pathogenesis of HIV Infection: No Progression with

Low-level Viremia

CD4

RNA

RNA Set Point ~ 103

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RNA

CD4

Pathogenesis: Average Progression with Median-Level Viremia

RNA Set Point ~104

Years

1 5 10

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Pathogenesis: Rapid Progression with High-

Level Viremia

RNA Set Point ~ 106

2 3

Years

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Direct detection of virus

Viral culture

Viral Load

DNA PCR

ANTIGEN (Ag) in plasma/serum (p24)

Detection of Antibody

Rapid tests

ELISA

Western blot

Laboratory Tests for Diagnosis

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Interpretation of Rapid HIV Testing


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An initial negative rapid test can be accepted as true

true negative = not HIV infected

NB. you need to consider that the patient may be in the

window period.

Positive rapid test ---Needs to be confirmed

Only if a positive test is confirmed, you report result to the patient!

Interpretation of Rapid HIV Testing

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Rapid Tests


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Rapid Tests

Various tests that provide results in ~10-20 minutes

Sensitivity approaches 100%; specificity is high as well

Negative tests can be reported as negatives

Positive results should be confirmed

Eth : Serial Testing Algorithm ( KHB, Statpk and Unigold)

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ELISA

Microplate ELISA for HIV antibody: coloured wells indicate

reactivity

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Western blots

Screening by ELISA followed by a confirmatory test

( WB).

WB: Abs to HIV-1 proteins

Core (p17,p24,p55), polymerase (p31, p51,p66 )and

envelop( gp 41, gp120, gp160)

Accuracy: > 3 months after transmission 99.5%

Sensitivity, 99.9% specificity

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Western blots

HIV-1 Western Blot

Lane1: Positive Control

Lane 2: Negative Control

Sample A: Negative

Sample B: Indeterminate

Sample C: Positive

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Acute HIV infection- High VL and Negative or

indeterminate HIV serology.

Recommended: Plasma HIV RNA VL

Viral Detection DNA ( HIV-1 DNA PCR)

DBS / whole blood) 100% sensitivity,96.6% specificity.

RNA ( HIV-1 RNA) by RT-PCRQuantitative Plasma HIV RNA ( VL)

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CD4 count

It is the most reliable indicator of prognosis

Important to maketherapeutic decisions. ( 200/ 350)

Analysis of 13 cohorts with 16,214 pts found that the CD4count was by far the most important predictor of death

CD8 cell counts have not been found to predict outcome

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CD4 count

Technique -- BD FACSCount, and pointCare technologies.

Baseline, repeated every 3 to 6 months.

Factors that influence CD4 cell counts: Analytical variation,Seasonal and diurnal variations ( lowest level 12:30pm and

peak values 8:30pm)

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CD4 count

Modest decreases in the CD4 cell count

Acute infections, major surgery, corticosteroid administration,

interferon treatment

Deceptively high CD4 counts: HTLV-1 co- infection or

splenectomy

CD4% may be better for predicting disease progression with

CD4 count >350/mm3

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Response to HAART

CD4 count increase by > 50cells/mm3 at 4 to 8 weeks.

One year averages 100- 150 cells/mm3 , at 3-5 years itaverages 20-50 cells /mm3 and at > 5 years it averages 20-30

cells/ mm3

Patients with baseline counts >350/mm3 had nearly normal

CD4 counts after 6 years of HAART with good viral suppression

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CD4 count as a surrogate for Virologic failure

Total Lymphocyte Count ( TLC) sometimes used as a

surrogate for CD4 count

TLC


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Therapy for HIV infection

Eradication of HIV infection cannot be achieved with currently

available ART regimens

therefore treatment to suppress the virus is lifelong.

Rx has resulted in substantial reductions in HIV-related

morbidity and mortality.

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HAART (Highly Active Antiretroviral Therapy)- available since 1995

Classes of anti-retroviral drugs:

1. Nucleoside / Nucleotide Reverse Transcriptase Inhibitors

(NRTI's) 2. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI's)

3. Protease Inhibitors (PI's)

4. Fusion inhibitors

5. Co-receptor binding inhibitor

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Prevention

Safe sex,

ABC

safe use of needles

Treat HIV-1 infected pregnant women to prevent infection of

the fetus/infant.

Vaccines: No vaccine is currently approved.

Clinical trials are currently being conducted with a number of

different vaccines 76


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Coreceptor, CD4Binding Inhibitors

maravirocvicrivirocTNX 355

Fusion Inhibitors

enfuvirtide

Reverse TranscriptaseInhibitors

Maturation Inhibitorbevirimat

saquinavir indinavir

ritonavir nelfinavir

fosamprenav

ir

lopinavir

atazanavir tipranavi

r

darunavir

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Diagnostics

Clinical

Services

Thank you

Documents

13. Retroviridae