Woro Hapsari W

What is HIV?
Human: Infecting human beings
Immunodeficiency: Decrease or weakness in
the bodys ability to fight off infections and
illnesses
Virus: A pathogen having the ability to
replicate only inside a living cell

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 HIV infects : variety of cells in the immune
system
CD4+ helper T cells
Macrophages
Dendritic cells
AIDS profound immunosuppression
Opportunistic infections
Malignant tumors
Wasting
CNS degeneration
 Adults and children estimated to be living with HIV
as of end 2005
Western & Central Eastern Europe
Europe & Central Asia
North America 720 000 1.6 million
1.2 million [570 000 890 000][990 000 2.3 million]East Asia
[650 000 1.8 million] 870 000
Caribbean North Africa & Middle East [440 000 1.4 million]
300 000 510 000
[230 000 1.4 million] South & South-East Asia
[200 000 510 000]
7.4 million
Sub-Saharan Africa [4.5 11.0 million]
Latin America 25.8 million Oceania
1.8 million [23.8 28.9 million]
74 000
[1.4 2.4 million]
[45 000 120 000]

Total: 40.3 (36.7 45.3) million

Source: UNAIDS. AIDS Epidemic Update 2005

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 Types of HIV Virus
HIV 1
Most common in sub-Saharan Africa and
throughout the world
Most common cause of AIDS

HIV 2
Most often found in West Central Africa, parts of
Europe and India
Slower progression

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HIV structure
Viral life cycle
Mechanisme of HIV entry into a cell
Pathogenesis of HIV infection & AIDS
Pathogenesis of HIV infection & AIDS
 Respon Imun terhadap HIV

Dipotong
HIV masuk ke Difagosit oleh
menjadi bagian
dalam tubuh makrofag
yang kecil2

Limfosit T mengenali Ag Potongan kecil2

(teraktivasi; membelah ditampilkan pada
dengan cepat) membran makrofag
 Respon Imun terhadap HIV

Limfosit T
helper

Sel T
Sel B
sitotoksik
 Respons Imun Non Spesifik
IFN menghambat replikasi virus
Sel NK mampu mebunuh virus yang berada di
dalam sel
Aktivasi komplemen & fagositosis eliminasi
virus yg dtg dari ekstraseluler & sirkulasi
Clinical course of HIV infection & AIDS
 Disease Progression
Severity of illness is determined by amount of
virus in the body (increasing viral load) and
the degree of immune suppression
(decreasing CD4+ counts)
As the CD4 count declines, the immune
function decreases.

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WHO HIV/AIDS Classification System
Stage
Stage II Stage
Stage IIII Stage
Stage III
III Stage
Stage IV
IV
Minor
Minor Symptoms
Symptoms Moderate
Moderate
Asymptomatic
Asymptomatic Symptoms
Symptoms AIDS
AIDS

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 Hubungan jumlah CD4 dengan Risiko Infeksi
Oportunistik
Kadar CD4 Infeksi oportunistik
Tanpa cut of Sarkoma Kaposis, tuberkulosis, infeksi herpes
zoster virus, pneumonia bakterial, limfoma non
Hodgkin

< 250 sel/ l Pneumosistis pneumonia, kandidiasis esofageal,

Progressive multifocal leukoencephalopathy, infeksi
Herpes Simplex Virus

<100 sel/ l Toksoplasmosis serebral, infeksi kriptokokus,

tuberkulosis milier

<50 sel/ l Retinitis sitomegalovirus, mikobakteriosis atipikal

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Jumlah CD4 dan Infeksi oportunistik

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Oral Candidiasis (thrush)
 Oral Hairy Leukoplakia

Being that HIV reduces immunologic activity, the intraoral

environment is a prime target for chronic secondary infections
and inflammatory processes, including OHL, which is due to
the Epstein-Barr virus under immunosuppressed conditions
 Kaposis sarcoma (KS)
Kaposis sarcoma (shown) is
a rare cancer of the blood
vessels that is associated
with HIV. It manifests as
bluish-red oval-shaped
patches that may eventually
become thickened. Lesions
may appear singly or in
clusters.
 How is HIV Transmitted?
Unprotected sexual contact
with an infected partner
Exposure of broken skin or
wound to infected blood or
body fluids
Transfusion with HIV-
infected blood
Injection with
contaminated objects
Mother to child during
pregnancy, birth or
breastfeeding
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 Infants with HIV
Failure to thrive
Persistent oral candidiasis
Hepatosplenomegaly
Lymphadenopathy
Recurrent diarrhea
Recurrent bacterial infections
Abnormal neurologic findings.
 Testing for Viral Infection and Immune
Response
Viral infection
Viral Load
p24 Antigen
Immune response
Antibody (IgG, IgM)
Cellular response (CD4)

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Immune response to HIV infection

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 Evolution of Antibodies

Window Period

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 typical primary HIV-1 infection
symptoms symptom
s
window HIV proviral DNA
period
HIV antibodies

HIV viral load

HIV-1 p24 antigen

0 1 2 3 4 5 6 / 2 4 6 8 10
1 infection weeks years
Time following infection
 Window Period
Time from initial infection with HIV until
antibodies are detected by a single test
Usually 3-8 weeks before antibodies are
detected
May test false-negative for HIV antibodies
during this time period
Can still pass the virus to others during this
period

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 Strategi Tes HIV Berdasarkan Tujuan dan Prevalensi Setempat
Tujuan tes Kondisi klinis Prevalensi Strategi
setempat
Donor darah Semua I
dan prevalensi
transplantasi
Surveilans >10% I

10% II

Diagnosis Simtomatik >10% II

Asimtomatik 10% III

Indonesia dengan prevalensi HIV di bawah 10% menggunakan strategi 3

& selalu didahului dengan konseling pra tes
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32
 Pemeriksaan Limfosit T CD4

FACS : Flowcytometry and Cell Sorting

Prinsip : Flowcytometry
Sel CD4 + FITC-Ab dilabel oleh fluorokrom dilewatkan satu
persatu ke chamber ditembakkan sinar laser
mengaktivasi fluorokrom analisis sinyal sel dipisahkan
berdasar muatan emisi cahaya ditangkap oleh
Photomultiplier tube amplifikasi sinyal intensitas
fluoresensi
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34
Kelebihan &
kekurangan
metode
pemeriksaan
CD4

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Clin Infect Dis. Vol 37 (2003)
 Laboratory Diagnosis of HIV Infection

Methods utilized to detect:

Antibody
Antigen
Viral nucleic acid
Virus in culture
 ELISA Testing
ELISA tests useful for:
Screening blood products.
Diagnosing and monitoring patients.
Determining prevalence of infection.
Research investigations.
 ELISA Testing
Different types of ELISA techniques used:
indirect
competitive
sandwich
ELISAs are for screening only, false positives do
occur and may be due to AI disease,
alcoholism, syphilis, and immunoproliferative
diseases.
ELISA Sandwich
4 generasi pemeriksaan anti-HIV metode
Enzyme Immuno Assay (EIA)
Generasi 1 mendeteksi antibodi IgG terhadap
HIV-1
Generasi 2 mendeteksi antibodi IgG terhadap
HIV-1 dan HIV-2
Generasi 3 mendeteksi antibodi IgM dan IgG
terhadap HIV-1 dan HIV-2
Generasi 4 mendeteksi antigen p24 dalam
sampel selain antibodi IgM dan IgG

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 Other Screening Tests
Agglutination tests using latex particles, gelatin
particles or microbeads are coated with HIV antigen
and will agglutinate in the presence of antibody.
Dot-Blot Testing utilizes paper or nitrocellulose
impregnated with antigen, patient serum is filtered
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.
Particle Agglutination
 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 tagged with
radioisotope or enzyme is added.
Specific bands form where antibody has reacted with
different antigens.
Most critical reagent of test is purest quality HIV antigen.
The following antigens must be present: p17, p24, p31,
gp41, p51, p55, p66, gp120 and gp160.
 Western Blot
Antibodies to p24 and p55 appear earliest but
decrease or become undetectable.
Antibodies to gp31, gp41, gp 120, and gp160
appear later but are present throughout all
stages of the disease.
 Western Blot
Interpretation of results.
No bands, negative.
In order to be interpreted as positive a minimum
of 3 bands directed against the following antigens
must be present: p24, p31, gp41 or gp120/160.
CDC criteria require 2 bands of the following:
p24, gp41 or gp120/160.
 gp160
gp120

p68
p55
p53

gp41-45

Spectrum p40

of anti-HIV
p34

p24

testing p18

p12

early recent / established advanced

DNA PCR
RNA PCR
p24 Ag
3rd gen ELISA
1st gen ELISA
Detuned ELISA
1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr
Western Blot
Expensive $ 80 - 100
technically more difficult
visual interpretation
lack standardisation
- performance
- interpretation
- indeterminate reactions
resolution of ??
Gold Standard for
confirmation
 Western Blot
Indeterminate results are those samples that produce bands
but not enough to be positive, may be due to the following:
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 and negative controls.
 Indirect immunofluorescence
Can be used to detect both virus and antibody
to it.
Antibody detected by testing patient serum
against antigen applied to a slide, incubated,
washed and a fluorescent antibody added.
Virus is detected by fixing patient cells to slide,
incubating with antibody.
 Detection of p24 HIV antigen
The p24-antigen screening assay is an EIA performed
on serum or plasma.
P24 antigen only present for short time, disappears
when antibody to p24 appears.
Anti-HIV-1 bound to membrane, incubated with
patient serum, second anti-HIV-1 antibody attached
to enzyme label is added (sandwich technique), color
change occurs.
Optical density measured, standard curve prepared
to quantitate results.
 Detection of p24 HIV antigen
Positive confirmed by neutralizing reaction,
preincubate patient sample with anti- HIV,
retest, if p24 present immune complexes form
preventing binding to HIV antibody on
membrane when added.
Test not recommended for routine screening
as appearance and rate of rise are
unpredictable.
Sensitivity lower than ELISA.
 Detection of p24 HIV antigen
Most useful for the following:
early infection suspected in seronegative patient
newborns
CSF
monitoring disease progress
 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 DNA sequences
present.
The DNA is detected by using radioactive or biotinylated
probes.
Once DNA is amplified it is placed on nitrocellulose paper and
allowed to react with a radiolabeled probe, a single stranded
DNA fragment unique to HIV, which will hybridize with the
patients HIV DNA if present.
Radioactivity is determined.
 Virus isolation
Virus isolation can be used to definitively diagnose
HIV.
Best sample is peripheral blood, but can use CSF,
saliva, cervical secretions, semen, tears or material
from organ biopsy.
Cell growth in culture is stimulated, amplifies number
of cells releasing virus.
Cultures incubated one month, infection confirmed
by detecting reverse transcriptase or p24 antigen in
supernatant.
 Viral Load Tests
Viral load or viral burden is the quantity of
HIV-RNA that is in the blood.
RNA is the genetic material of HIV that
contains information to make more virus.
 Viral Load Tests
Viral load tests measure the amount of HIV-RNA in
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 changing
antiretroviral therapy to determine effect on viral
load.
 Testing of Neonates
Difficult due to presence of maternal IgG
antibodies.
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 not
detecting antigen soon enough: 38 days to 6
months to be positive.
Di bawah ini adalah pemeriksaan laboratorium yang ideal
sebelum memulai ART apabila sumber daya memungkinkan:

Darah lengkap
Jumlah CD4
SGOT / SGPT
Kreatinin Serum
Urinalisa
HbsAg
Anti-HCV
Profil lipid serum
Gula darah
VDRL/TPHA/PRP
Ronsen dada (utamanya bila curiga ada infeksi paru)
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Pemantauan Pasien dalam Terapi Antiretroviral

1. Pemantauan klinis

Frekuensi tergantung respon terapi ARV.

Minimal minggu 2, 4, 8, 12 dan 24 minggu
sejak memulai terapi ARV dan kemudian
setiap 6 bulan bila pasien telah mencapai
keadaan stabil
Pada setiap kunjungan perlu dilakukan
penilaian klinis termasuk tanda dan gejala efek
samping obat atau gagal terapi dan frekuensi
infeksi (infeksi bakterial, kandidiasis dan atau
infeksi oportunistik lainnya) 61
 2. Pemantauan laboratoris

Pemantauan CD4 secara rutin setiap 6 bulan, atau lebih

sering bila ada indikasi klinis

Untuk pasien yang akan memulai terapi kadar

Hemoglobin (Hb) sebelum memulai terapi & minggu ke 4,
8 dan 12 sejak mulai terapi atau ada indikasi tanda dan
gejala anemia

Pengukuran ALT (SGPT) fungsi ginjal, kimia darah lainnya

perlu dilakukan bila ada tanda dan gejala dan bukan
berdasarkan sesuatu yang rutin
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 Pengukuran Viral Load (VL) sampai sekarang
tidak dianjurkan untuk memantau pasien dalam
terapi ARV dalam keadaan terbatas fasilitas dan
kemampuan pasien

Jika pengukuran VL dapat dilakukan maka terapi

ARV diharapkan menurunkan VL menjadi tidak
terdeteksi (undetectable) setelah bulan ke 6

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Can Disease Progression Be Delayed?

Prevention and early

treatment of
opportunistic
YES infections (OIs)
Antiretroviral therapy
Positive living

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Skema Interpretasi Strategi Diagnosis WHO untuk Infeksi HIV

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