Upload
dev-ashwani
View
215
Download
0
Embed Size (px)
Citation preview
7/28/2019 ser hiv aids
1/63
7/28/2019 ser hiv aids
2/63
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)
7/28/2019 ser hiv aids
3/63
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/
7/28/2019 ser hiv aids
4/63
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.
7/28/2019 ser hiv aids
5/63
Characteristics of the virus
7/28/2019 ser hiv aids
6/63
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).
7/28/2019 ser hiv aids
7/63
Structural Genes
Three main structural genes:
Group Specific Antigen (Gag)
Envelope (Env)
Polymerase (Pol)
7/28/2019 ser hiv aids
8/63
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.
7/28/2019 ser hiv aids
9/63
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.
7/28/2019 ser hiv aids
10/63
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.
7/28/2019 ser hiv aids
11/63
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.
7/28/2019 ser hiv aids
12/63
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
7/28/2019 ser hiv aids
13/63
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.html7/28/2019 ser hiv aids
14/63
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.
7/28/2019 ser hiv aids
15/63
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.
7/28/2019 ser hiv aids
16/63
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.html7/28/2019 ser hiv aids
17/63
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.
7/28/2019 ser hiv aids
18/63
7/28/2019 ser hiv aids
19/63
HIV (arrows) Infecting a T-lymphocyte
7/28/2019 ser hiv aids
20/63
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.
7/28/2019 ser hiv aids
21/63
Transmission
7/28/2019 ser hiv aids
22/63
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
7/28/2019 ser hiv aids
23/63
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.
7/28/2019 ser hiv aids
24/63
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)
7/28/2019 ser hiv aids
25/63
Oral Candidiasis (thrush)
7/28/2019 ser hiv aids
26/63
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
7/28/2019 ser hiv aids
27/63
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.
7/28/2019 ser hiv aids
28/63
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.
7/28/2019 ser hiv aids
29/63
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
7/28/2019 ser hiv aids
30/63
Infants with HIV
Failure to thrive
Persistent oral candidiasis
Hepatosplenomegaly Lymphadenopathy
Recurrent diarrhea
Recurrent bacterial infections Abnormal neurologic findings.
7/28/2019 ser hiv aids
31/63
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.
7/28/2019 ser hiv aids
32/63
7/28/2019 ser hiv aids
33/63
Immunologic Manifestations
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.
7/28/2019 ser hiv aids
34/63
Immunologic Manifestations
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.
7/28/2019 ser hiv aids
35/63
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
7/28/2019 ser hiv aids
36/63
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.
7/28/2019 ser hiv aids
37/63
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
7/28/2019 ser hiv aids
38/63
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
7/28/2019 ser hiv aids
39/63
Laboratory Diagnosis of HIV Infection
Methods utilized to detect:
Antibody
Antigen
Viral nucleic acid
Virus in culture
7/28/2019 ser hiv aids
40/63
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
7/28/2019 ser hiv aids
41/63
ELISA Testing
ELISA tests useful for:
Screening blood products.
Diagnosing and monitoring patients.
Determining prevalence of infection.
Research investigations.
7/28/2019 ser hiv aids
42/63
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.
7/28/2019 ser hiv aids
43/63
ELISA Sandwich
7/28/2019 ser hiv aids
44/63
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.
7/28/2019 ser hiv aids
45/63
Particle Agglutination
7/28/2019 ser hiv aids
46/63
7/28/2019 ser hiv aids
47/63
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.
7/28/2019 ser hiv aids
48/63
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.
7/28/2019 ser hiv aids
49/63
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
7/28/2019 ser hiv aids
50/63
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
7/28/2019 ser hiv aids
51/63
Western Blot
Expensive $ 80 - 100
technically more difficult
visual interpretation
lack standardisation - performance - interpretation
- indeterminate reactionsresolution of ??
Gold Standard forconfirmation
7/28/2019 ser hiv aids
52/63
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.
7/28/2019 ser hiv aids
53/63
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.
7/28/2019 ser hiv aids
54/63
7/28/2019 ser hiv aids
55/63
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.
7/28/2019 ser hiv aids
56/63
Detection of p24 HIV antigen
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.
7/28/2019 ser hiv aids
57/63
Detection of p24 HIV antigen
Most useful for the following:
early infection suspected in seronegativepatient
newborns CSF
monitoring disease progress
7/28/2019 ser hiv aids
58/63
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.
7/28/2019 ser hiv aids
59/63
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.
7/28/2019 ser hiv aids
60/63
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.
7/28/2019 ser hiv aids
61/63
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.
7/28/2019 ser hiv aids
62/63
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.
7/28/2019 ser hiv aids
63/63
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