ser hiv aids

7/28/2019 ser hiv aids

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Introduction

Etiologic agent of AcquiredImmunodeficiency Syndrome (AIDS).

Discovered independently by Luc

Montagnier of France and Robert Gallo ofthe US in 1983-84.

Former names of the virus include:

Human T cell lymphotrophic virus (HTLV-III) Lymphadenopathy associated virus (LAV)

AIDS associated retrovirus (ARV)


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Introduction

HIV-2 discovered in 1986, antigenicallydistinct virus endemic in West Africa.

One million people infected in US, 30million worldwide are infected.

Leading cause of death of men aged 25-44 and 4th leading cause of death ofwomen in this age group in the US.

http://www.cnn.com/2005/HEALTH/conditions/11/17/blacks.hiv.ap/


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Characteristics of the virus

Icosahedral (20 sided), enveloped virus of thelentivirus subfamily ofretroviruses.

Retroviruses transcribe RNA to DNA.

Two viral strands of RNA found in coresurrounded by protein outer coat.

Outer envelope contains a lipid matrix within whichspecific viral glycoproteins are imbedded.

These knob-like structures responsible for binding totarget cell.


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Characteristics of the virus


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HIV

The outer shell of the virus isknown as the Viral enevlope.Embedded in the viralenvelope is a complex proteinknown as envwhich consists

of an outer protruding capglycoprotein (gp) 120, and astem gp14. Within the viralenvelope is an HIV proteincalledp17(matrix), and withinthis is the viral core or capsid,

which is made of another viralprotein p24(core antigen).


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Structural Genes

Three main structural genes:

Group Specific Antigen (Gag)

Envelope (Env)

Polymerase (Pol)


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Group Specific Antigen (Gag)

Located in nucelocapsid of virus.

Icosahedryl capsid surrounds the internalnucleic acids made up ofp24 andp15.

p17lies between protein core andenvelope and is embedded in the internalportion of the envelope.

Two additionalp55products,p7andp9,are nucleic acid binding proteins closelyassociated with the RNA.


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Envelope (Env)

Envelope (Env) gene codes for envelopeproteins gp160, gp120and gp41. These polyproteins will eventually be cleaved by

proteases to become HIV envelope glycoproteins

gp120 and gp41. gp160cleaved to form gp120and gp41. gp120forms the 72 knobs which protrude from outer

envelope. gp41 is a transmembrane glycoprotein antigen that

spans the inner and outer membranes and attachesto gp120. gp120and gp41 both involved with fusion and

attachment of HIV to CD4 antigen on host cells.


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Polymerase (Pol)

Polymerase (Pol) codes for p66 and p51subunits of reverse transcriptase and p31an endonuclease.

Located in the core, close to nucleic acids.

Responsible for conversion of viral RNA intoDNA, integration of DNA into host cell DNA

and cleavage of protein precursors.


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Viral Replication

First step, HIV attaches to susceptible host cell.

Site of attachment is the CD4 antigen found on avariety of cells

helper T cells macrophages

monocytes

B cells

microglial brain cells

intestinal cells

T cells infected later on.


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Early Phase HIV Infection

In early phase HIVinfection, initialviruses are M-tropic.

Their envelopeglycoprotein gp120 isable to bind to CD4molecules and

chemokine receptorscalled CCR5 found onmacrophages


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http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivad.html

In late phase HIVinfection, most of theviruses are T-tropic,

having gp120 capableof binding to CD4 andCXCR4 found on T4-lymphocytes.
http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivad.htmlhttp://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivad.html


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Life Cycle

(a) HIV (red) attaches to two cell-surface receptors(the CD4 antigen and a specific chemokinereceptor).

(b) The virus and cell membrane fuse, and thevirion core enters the cell.

(c) The viral RNA and core proteins are releasedfrom the virion core and are then activelytransported to the nucleus.

(d) The viral RNA genome is converted into double-stranded DNA through an enzyme unique toviruses, reverse transcriptase (red dot).

(e) The double-stranded viral DNA moves into thecell nucleus.

(f) Using a unique viral enzyme called integrase, theviral DNA is integrated into the cellular DNA.

(g) Viral RNA is synthesized by the cellular enzymeRNA polymerase II using integrated viral DNA as atemplate. Two types of RNA transcripts shorterspliced RNA (h) and full-length genomic RNA (j) areproduced.

(h) Shorter spliced RNAs are transported to thecytoplasm and used for the production of severalviral proteins that are then modified in the Golgiapparatus of the cell (i).

(j) Full-length genomic RNAs are transported to thecytoplasm (k).

(l) New virion is assembled and then buds off. (m) Mature virus is released.


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Viral Replication

The gp120 protein on virus bindsspecifically to CD4 receptor on host cellwith high affinity.

Gp41 causes fusion of the virus to the cellmembrane.

After fusion virus particle enters cell.

Viral genome exposed by uncoating particle.


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Viral Replication

Reverse transcriptase produces viral DNAfrom RNA.

Becomes a provirus which integrates into host

DNA.

Period of latency occurs. http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivdsdna.html
http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivdsdna.htmlhttp://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivdsdna.html


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Viral Replication

After a period of latency lasting up to 10 yearsviral replication is triggered and occurs at highrate.

CD4 cell may be destroyed in the process, bodyattempts to replace lost CD4 cells, but over thecourse of many years body is unable to keep thecount at a safe level.

Destruction of large numbers of CD4 causesymptoms of HIV to appear with increasedsusceptibility to opportunistic infections, diseaseand malignancy.


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HIV (arrows) Infecting a T-lymphocyte


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Viral Replication

Methods of transmission:

Sexual transmission, presence of STD increaseslikelihood of transmission.

Exposure to infected blood or blood products. Use of contaminated clotting factors by hemophiliacs.

Sharing contaminated needles (IV drug users).

Transplantation of infected tissues or organs.

Mother to fetus, perinatal transmission variable,dependent on viral load and mothers CD 4 count.


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Transmission


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Primary HIV Syndrome

Mononucleosis-like, cold or flu-like symptomsmay occur 6 to 12 weeks after infection. lymphadenopathy

fever

rash

headache

Fatigue

diarrhea

sore throat

neurologic manifestations.

no symptoms may be present


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Primary HIV Syndrome

Symptoms are relatively nonspecific.

HIV antibody test often negative but becomespositive within 3 to 6 months, this process is

known as seroconversion. Large amount of HIV in the peripheral blood.

Primary HIV can be diagnosed using viral loadtiter assay or other tests.

Primary HIV syndrome resolves itself and HIVinfected person remains asymptomatic for aprolonged period of time, often years.


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Clinical Latency Period

HIV continues to reproduce, CD4 countgradually declines from its normal value of 500-1200.

Once CD4 count drops below 500, HIV infected

person at risk foropportunistic infections. The following diseases are predict iveof the

progression to AIDS: persistent herpes-zoster infection (shingles)

oral candidiasis (thrush) oral hairy leukoplakia Kaposis sarcoma (KS)


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Oral Candidiasis (thrush)


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Oral Hairy Leukoplakia

Being that HIV reduces immunologic activity, theintraoral environment is a prime target for chronic

secondary infections and inflammatory processes,including OHL, which is due to the Epstein-Barr virusunder immunosuppressed conditions


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Kaposis sarcoma (KS)

Kaposis sarcoma

(shown) is a rare cancerof the blood vessels thatis associated with HIV. It

manifests as bluish-redoval-shaped patches thatmay eventually becomethickened. Lesions may

appear singly or inclusters.


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AIDS

CD4 count drops below 200person is considered tohave advanced HIV disease

If preventative medications not started the HIV infectedperson is now at risk for: Pneumocystis carinii pneumonia (PCP) cryptococcal meningitis toxoplasmosis

If CD4 count drops below 50: Mycobacterium avium Cytomegalovirus infections

lymphoma dementia Most deaths occur with CD4 counts below 50.


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Other Opportunistic Infections

Respiratory system Pneumocystis Carinii Pneumonia (PCP) Tuberculosis (TB) Kaposi's Sarcoma (KS)

Gastro-intestinal system Cryptosporidiosis Candida Cytomegolavirus (CMV)

Isosporiasis Kaposi's Sarcoma

Central/peripheral Nervous system Cytomegolavirus Toxoplasmosis Cryptococcosis Non Hodgkin's lymphoma Varicella Zoster Herpes simplex

Skin Herpes simple Kaposi's sarcoma Varicella Zoster


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Infants with HIV

Failure to thrive

Persistent oral candidiasis

Hepatosplenomegaly Lymphadenopathy

Recurrent diarrhea

Recurrent bacterial infections Abnormal neurologic findings.


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Immunologic Manifestations

Early stage slight depression of CD4count, few symptoms, temporary.

Window of up to 6 weeks before antibodyis detected, by 6 months 95% positive.

During window p24 antigen present, acuteviremia and antigenemia.


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Immune abnormalities associated with increasedviral replication. Decrease in CD4 cells due to virus budding from

cells, fusion of uninfected cells with virally infected

cells and apoptosis. B cells have decreased response to antigens possiblydue to blockage of T cell/B cell interaction by bindingof viral proteins to CD4 site.

CD8 cells initially increase and may remain elevated.

As HIV infection progresses, CD4 T cells dropresulting in immunosuppression and susceptibility ofpatient to opportunistic infections.

Death comes due to immuno-incompetence.


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Immune abnormalities associated with increasedviral replication. Decrease in CD4 cells due to virus budding from

cells, fusion of uninfected cells with virally infected

cells and apoptosis. B cells have decreased response to antigens possiblydue to blockage of T cell/B cell interaction by bindingof viral proteins to CD4 site.

CD8 cells initially increase and may remain elevated.

As HIV infection progresses, CD4 T cells dropresulting in immunosuppression and susceptibility ofpatient to opportunistic infections.

Death comes due to immuno-incompetence.


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The Move Toward Lower Pill BurdensDosing Daily pill burdenRegimen

1996

Zerit/Epivir/Crixivan 10 pills, Q8H

20023 pills, BIDCombivir (AZT/3TC)/EFV

1998Retrovir/Epivir/Sustiva 5 pills, BID

2003

3 pills, QDViread/ Emtriva/Sustiva

2004

2 pills, QDTruvada/Sustiva


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Sustiva + Truvada Treatment

Sustiva + Truvada (FTC + tenofovor) is one of the mostpopular and effective starting HIV regimens.

Many patients will have dream/sleep/central nervoussystem effects particularly in the first month (due to theSustiva).

Upset stomach/bloating/gas/loose stools is also fairlycommon during the first month and for most patients isfairly mild.

HIV levels in the blood will often drop by > 99% in thefirst month and the CD4 count (marker of immune

system function) will often increase providing protectionagainst AIDS related diseases within weeks/months ofstarting the medication.


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Truvada

Truvada is made up ofHIV drugs from aclass called nucleoside/nucleotide reversetranscriptase inhibitors (NRTIs), also

known as nukes.

The NRTIs block reverse transcriptase, aprotein that HIV needs to make more

copies of itself. This may slow down HIVdisease


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typical primary HIV-1 infection

symptoms

HIV-1 p24 antigen

0 1 2 3 4 5 6 / 2 4 6 8 10

weeks years

HIV antibodies

Time following infection

HIV viral load

HIV proviral DNA

symptoms

window

period

1 infection


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Laboratory Diagnosis of HIV Infection

Methods utilized to detect:

Antibody

Antigen

Viral nucleic acid

Virus in culture


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ELISA Testing

First serological test developed to detectHIV infection. Easy to perform.

Easily adapted to batch testing. Highly sensitive and specific.

Antibodies detected in ELISA include

those directed against: p24, gp120, gp160and gp41, detected first in infection andappear in most individuals


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ELISA Testing

ELISA tests useful for:

Screening blood products.

Diagnosing and monitoring patients.

Determining prevalence of infection.

Research investigations.


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ELISA Testing

Different types of ELISA techniques used:

indirect

competitive

sandwich

ELISAs are for screening only, falsepositives do occur and may be due to AI

disease, alcoholism, syphilis, andimmunoproliferative diseases.


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ELISA Sandwich


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Other Screening Tests

Agglutination tests using latex particles, gelatinparticles or microbeads are coated with HIVantigen and will agglutinate in the presence of

antibody. Dot-Blot Testing utilizes paper or nitrocellulose

impregnated with antigen, patient serum isfiltered through, and anti-antibody is added with

enzyme label, color change is positive. A rapid, cost-effective and may become an alternative

to standard ELISA and Western blot testing.


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Particle Agglutination


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Western Blot

Most popular confirmatory test. Utilizes a lysate prepared from HIV virus. The lysate is electrophoresed to separate out the HIV

proteins (antigens).

The paper is cut into strips and reacted with test sera. After incubation and washing anti-antibody taggedwith radioisotope or enzyme is added.

Specific bands form where antibody has reacted withdifferent antigens.

Most critical reagent of test is purest quality HIVantigen. The following antigens must be present: p17, p24,

p31, gp41, p51, p55, p66, gp120 and gp160.


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Western Blot

Antibodies to p24 and p55 appear earliestbut decrease or become undetectable.

Antibodies to gp31, gp41, gp 120, and

gp160 appear later but are presentthroughout all stages of the disease.


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Western Blot

Interpretation of results.

No bands, negative.

In order to be interpreted as positive a

minimum of 3 bands directed against thefollowing antigens must be present: p24, p31,gp41 or gp120/160.

CDC criteria require 2 bands of thefollowing: p24, gp41 or gp120/160.

gp160gp160gp160


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DNA PCR

RNA PCRp24 Ag

3rd gen ELISA

1st gen ELISA

Detuned ELISA1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr

gp160

gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160

gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160

gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

early recent / established advanced

Spectrum

of anti-HIV

testing


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Western Blot

Expensive $ 80 - 100

technically more difficult

visual interpretation

lack standardisation - performance - interpretation

- indeterminate reactionsresolution of ??

Gold Standard forconfirmation


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Western Blot

Indeterminate results are those samples that producebands but not enough to be positive, may be due to thefollowing: prior blood transfusions, even with non-HIV-1 infected blood

prior or current infection with syphilis

prior or current infection with malaria

autoimmune diseases (e.g., diabetes, Graves disease, etc)

infection with other human retroviruses

second or subsequent pregnancies in women.

run an alternate HIV confirmatory assay. Quality control of Western Blot is critical and requires

testing with strongly positive, weakly positive andnegative controls.


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Indirect immunofluorescence

Can be used to detect both virus andantibody to it.

Antibody detected by testing patient serum

against antigen applied to a slide,incubated, washed and a fluorescentantibody added.

Virus is detected by fixing patient cells toslide, incubating with antibody.


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Detection of p24 HIV antigen

The p24-antigen screening assay is an EIAperformed on serum or plasma.

P24 antigen only present for short time,disappears when antibody to p24 appears.

Anti-HIV-1 bound to membrane, incubated withpatient serum, second anti-HIV-1 antibodyattached to enzyme label is added (sandwichtechnique), color change occurs.

Optical density measured, standard curveprepared to quantitate results.


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Positive confirmed by neutralizingreaction, preincubate patient sample withanti- HIV, retest, if p24 present immune

complexes form preventing binding to HIVantibody on membrane when added.

Test not recommended for routinescreening as appearance and rate of rise

are unpredictable. Sensitivity lower than ELISA.


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Most useful for the following:

early infection suspected in seronegativepatient

newborns CSF

monitoring disease progress


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Polymerase Chain Reaction (PCR)

Looks for HIV DNA in the WBCs of a person. PCR amplifies tiny quantities of the HIV DNA present,

each cycle of PCR results in doubling of the DNAsequences present.

The DNA is detected by using radioactive or biotinylatedprobes.

Once DNA is amplified it is placed on nitrocellulosepaper and allowed to react with a radiolabeled probe, asingle stranded DNA fragment unique to HIV, which will

hybridize with the patients HIV DNA if present. Radioactivity is determined.


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Virus isolation

Virus isolation can be used to definitivelydiagnose HIV.

Best sample is peripheral blood, but can useCSF, saliva, cervical secretions, semen, tears ormaterial from organ biopsy.

Cell growth in culture is stimulated, amplifiesnumber of cells releasing virus.

Cultures incubated one month, infectionconfirmed by detecting reverse transcriptase orp24 antigen in supernatant.


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Viral Load Tests

Viral load or viral burden is the quantity ofHIV-RNA that is in the blood.

RNA is the genetic material of HIV that

contains information to make more virus.


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Viral Load Tests

Viral load tests measure the amount of HIV-RNAin one milliliter of blood.

Take 2 measurements 2-3 weeks apart to

determine baseline. Repeat every 3-6 months in conjunction with

CD4 counts to monitor viral load ant T-cell count.

Repeat 4-6 weeks after starting or changingantiretroviral therapy to determine effect on viralload.


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Testing of Neonates

Difficult due to presence of maternal IgGantibodies.

Use tests to detect IgM or IgA antibodies,

IgM lacks sensitivity, IgA more promising.

Measurement of p24 antigen.

PCR testing may be helpful but still notdetecting antigen soon enough: 38 days to6 months to be positive.


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References

http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivlc.html#translat

http://pathmicro.med.sc.edu/lecture/HIV3.htm

http://www.avert.org/hivstages.htm

http://www.aidsinfo.nih.gov/guidelines/ http://www.hopkins-aids.edu/publications/pocketguide/pocketgd0105.pdf

http://www.modares.ac.ir/sci/saman_h/Pages/applications.htm

http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02

http://www.hivandhepatitis.com/recent/test/realtime/061604_f.html
http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivlc.htmlhttp://pathmicro.med.sc.edu/lecture/HIV3.htmhttp://www.avert.org/hivstages.htmhttp://www.aidsinfo.nih.gov/guidelines/http://www.hopkins-aids.edu/publications/pocketguide/pocketgd0105.pdfhttp://www.modares.ac.ir/sci/saman_h/Pages/applications.htmhttp://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://www.hivandhepatitis.com/recent/test/realtime/061604_f.htmlhttp://www.hivandhepatitis.com/recent/test/realtime/061604_f.htmlhttp://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02http://www.modares.ac.ir/sci/saman_h/Pages/applications.htmhttp://www.hopkins-aids.edu/publications/pocketguide/pocketgd0105.pdfhttp://www.hopkins-aids.edu/publications/pocketguide/pocketgd0105.pdfhttp://www.hopkins-aids.edu/publications/pocketguide/pocketgd0105.pdfhttp://www.aidsinfo.nih.gov/guidelines/http://www.avert.org/hivstages.htmhttp://pathmicro.med.sc.edu/lecture/HIV3.htmhttp://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivlc.html

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