Disusun oleh :

Dr. H. Deddy Hartanto

Etiologi virus dari Acquired Immunodeficiency Syndrome (AIDS).

ADA 2 jenis virus HIV : HIV-1 dan HIV-2 HIV -1ditemukan oleh Luc Montagnier

of France dan Robert Gallo dari USA in 1983-84.

Nama-nama lain dari virus HIV:Human T cell lymphotrophic virus (HTLV-III)Lymphadenopathy associated virus (LAV)AIDS associated retrovirus (ARV)

HIV -1 menyebar lebih banyak daripada HIV- 2 di seluruh dunia.

HIV-2 ditemukan th 1986, antigen virus menyebar scr endemis di Afrika barat.

1 juta orang terinfeksi di USA, menyebar 30 juta orang seluruh dunia terinfeksi (2005).

Penyebab kematian utama pada laki2 umur 25-44 th dan penyebab ke 4 kematian wanita di USA.

Icosahedral (20 sisi), enveloped virus, subfamili lentivirus dari retroviruses.

Retroviruses menyalin RNA ke DNA. 2 helai virus dari RNA ditemukan

dalam inti yg dikelilingi oleh pelapis luar protein.Outer envelope contains a lipid matrix

within which specific viral glycoproteins are imbedded.

These knob-like structures responsible for binding to target cell.

The outer shell of the virus is known as the Viral enevlope. Embedded in the viral envelope is a complex protein known as env which consists of an outer protruding cap glycoprotein (gp) 120, and a stem gp14. Within the viral envelope is an HIV protein called p17(matrix), and within this is the viral core or capsid, which is made of another viral protein p24(core antigen).

Three main structural genes:Group Specific Antigen (Gag)Envelope (Env)Polymerase (Pol)

Located in nucelocapsid of virus. Icosahedryl capsid surrounds the

internal nucleic acids made up of p24 andp15.

p17 lies between protein core and envelope and is embedded in the internal portion of the envelope.

Two additional p55 products, p7 and p9, are nucleic acid binding proteins closely associated with the RNA.

Envelope (Env) gene codes for envelope proteins gp160, gp120 and gp41.These polyproteins will eventually be

cleaved by proteases to become HIV envelope glycoproteins gp120 and gp41.

gp160 cleaved to form gp120 and gp41.gp120 forms the 72 knobs which protrude

from outer envelope.gp41 is a transmembrane glycoprotein

antigen that spans the inner and outer membranes and attaches to gp120.

gp120 and gp41 both involved with fusion and attachment of HIV to CD4 antigen on host cells.

Polymerase (Pol) codes for p66 and p51 subunits of reverse transcriptase and p31 an endonuclease.Located in the core, close to nucleic acids.Responsible for conversion of viral RNA into

DNA, integration of DNA into host cell DNA and cleavage of protein precursors.

First step, HIV attaches to susceptible host cell.Site of attachment is the CD4 antigen

found on a variety of cells helper T cells macrophages monocytes B cells microglial brain cells intestinal cells

T cells infected later on.

In early phase HIV infection, initial viruses are M-tropic. Their envelope glycoprotein gp120 is able to bind to CD4 molecules and chemokine receptors called CCR5 found on macrophages

In late phase HIV infection, most of the viruses are T-tropic, having gp120 capable of binding to CD4 and CXCR4 found on T4-lymphocytes.

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

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

(c) The viral RNA and core proteins are released from the virion core and are then actively transported to the nucleus.

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

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

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

(g) Viral RNA is synthesized by the cellular enzyme RNA polymerase II using integrated viral DNA as a template. Two types of RNA transcripts shorter spliced RNA (h) and full-length genomic RNA (j) are produced.

(h) Shorter spliced RNAs are transported to the cytoplasm and used for the production of several viral proteins that are then modified in the Golgi apparatus of the cell (i).

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

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

The gp120 protein on virus binds specifically to CD4 receptor on host cell with high affinity.

Gp41 causes fusion of the virus to the cell membrane.After fusion virus particle enters cell.Viral genome exposed by uncoating

particle.

Reverse transcriptase produces viral DNA from RNA.Becomes a provirus which integrates into

host DNA.Period of latency occurs.

After a period of latency lasting up to 10 years viral replication is triggered and occurs at high rate.

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

Destruction of large numbers of CD4 cause symptoms of HIV to appear with increased susceptibility to opportunistic infections, disease and malignancy.

Transmisi Penularan :Sexual transmission, presence of STD

increases likelihood 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 mother’s CD 4 count.

spt infeksi Mononucleosis-like, gejala spt batuk pilek dapat timbul 6 -12 weeks setelah infeksi : lymphadenopathyDemamRash ( bercak kemerahan di tubuh )Pusing LelahDIareSakit tenggorokamManifestasi gangguan syaraf Kadang2 tanpa gejala yg jelas.

Gejala relatif tidak spesifik. Test Anti HIV sering negatif tetapi

bisa menjadi positif 3- 6 bulan kemudian, proses ini dinamakan “ serokonversi”.

Jumlah yang besar dari HIV banyak didapatkan di darah perifer.

HIV pada fase primer ini dapat didiagnosa menggunakan pemeriksaan virus load titer atau test yang lain.

Orang yang terinfeksi HIV pada fase primer bisa tanpa gejala selama waktu yang lama, sering bertahun-tahun.

HIV berkelanjutan berproduksi, jumlah CD4 secara bertahap menurun dari jumlah normalnya yaitu 500-1200.

Sewaktu CD4 jumlahnya menurun dari 500, orang yg terinfeksi HIV mempunyai resiko terkena infeksi2 opportunistik dari luar.

Berikut ini adalah penyakit2 yang diprediksi mengarah progresivitas kearah AIDS:persistent herpes-zoster infection

(shingles)oral candidiasis (thrush)oral hairy leukoplakiaKaposi’s sarcoma (KS)

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

Kaposi’s 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.

Dihitung CD4 menurun <200 maka orang ini dipertimbangkan mendapatkan penyakit HIV yg tahap lanjut kearah AIDS.

Bila pencegahan secara medis tidak dimulai maka infeksi HIV mempunyai resiko untuk: Pneumocystis carinii pneumonia (PCP) cryptococcal meningitis toxoplasmosis

Bila dihitung CD4 < 50: Mycobacterium avium Cytomegalovirus infections lymphoma dementia Banyak terjadi kematian padaCD4 < 50.

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

Gangguan perkembangan Jamur mulut yg persistent Hepato megali Pembesaran KGB Diare berulang Infeksi bakteri berulang ditemukan gangg.syaraf.

Early stage slight depression of CD4 count, few symptoms, temporary.

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

During window p24 antigen present, acute viremia and antigenemia.

Antibodies produced to all major antigens.First antibodies detected produced against

gag proteins p24 and p55.Followed by antibody to p51, p120 and

gp41As disease progresses antibody levels

decrease.

Immune abnormalities associated with increased viral 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 possibly due to blockage of T cell/B cell interaction by binding of viral proteins to CD4 site.

CD8 cells initially increase and may remain elevated.

As HIV infection progresses, CD4 T cells drop resulting in immunosuppression and susceptibility of patient to opportunistic infections.

Death comes due to immuno-incompetence.

Target cell adalah T.helper (CD4+) Normal :CD4+ : 65 % dan CD8+ : 35% dari

total T-cell Pada penderita AIDS konsentrasi CD4+

menurun secara drastis. Konsentrasi CD4+ dihubungkan dengan

konsentrasi CD8 . Menyebabkan rasio CD4+ / CD8 terbalik

pada HIV / AIDS

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

20033 pills, QDViread/ Emtriva/Sustiva

20042 pills, QDTruvada/Sustiva

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

Menggunakan metode yg mendeteksi:AntibodyAntigenViral nucleic acid

Kultur Virus

TENTANG STANDAR PELAYANAN LABORATORIUM KESEHATAN

PEMERIKSA HIV DAN INFEKSI OPORTUNISTIK

Tentang Penggunaan strategi III : Kombinasi 3 reagen rapid test HIV → Tujuan Diagnosis.

Strategi III dengan persyaratan reagensia sebagai berikut : 1. Sensitivitas reagen pertama >99% 2. Spesifisitas reagen kedua > 98% 3. Spesifitas reagen ketiga > 95%

Untuk tujuan surveilans : reagen pertama harus memiliki sensitiviras >99%.

Spesifisitas reagen kedua >98%. Semua regensia yang dipakai harus

terdaftar di DepKes RI

Pemeriksaan anti-HIV disertai informed consent tertulis,

sebelumnya didahului dengan konseling pra-uji / test dan

sesudahnya konseling pasca-uji / test Pelaporan pemeriksaan dilaporkan reaktif dan non reaktif. KERAHASIAAN!!!!

test serologi awal yg dikembangkan untuk mendeteksi Infeksi HIV :Mudah digunakan.Mudah adaptasi utk test batch.Sensitivitas dan spesifisitasnya tinggi.

Antibodi2 dideteksi oleh ELISA termasuk yang melawan antigen : p24, gp120, gp160 and gp41, Terdeteksi utk infeksi awal dan muncul pada semua orang yg terinfeksi.

ELISA test digunakan utk:Screening product2 darah.Diagnosis dan monitoring patients.Menentukan prevalensi dari infeksiPenelitian.

Ada 3 teknik metode yang digunakan: indirectcompetitivesandwich

ELISAs hanya utk penyaring saja,, positives palsu dapat timbul dan mungkin bisa terjadi pada AI disease, alcoholism, syphilis, and immunoproliferative diseases.

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.

Test Konfirmasi yang paling populer :Memanfaatkan preparasi lysate dari HIV

virus. lysate adalah electrophoresis yang

digunakan utk memisahkan keluar dari protein HIV (antigens).

Kertas dipotong kedalam strips dan direaksikan dengan sera test.

Setelah inkubasi dan pencucian anti antibodi dengan ditambah radioisotop atau enzym.

Pita2 spesifik dimana antibodi direaksikan dengan antigen2 yang berbeda.

Reagen yang terbanyak dari test ini adalah yang paling murni kualitas antigen HIV nya.

Antigen2 yang harus ada dalam pemeriksaan sbb: p17, p24, p31, gp41, p51, p55, p66, gp120 and gp160.

Antibodi terhadap p24 dan p55 muncul lebih awal tetapi jumlah menurun dan menjadi tidak ditemukan.

Antibodi terhadap gp31, gp41, gp 120, and gp160 muncul belakangan tetapi selalu ada pada setiap tingkat dari penyakit.

DNA PCRDNA PCRRNA PCRRNA PCR

p24 Agp24 Ag3rd gen ELISA1st gen ELISA

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

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

gp160gp120

p68p55p53

gp41-45

p40

p34

p24

p18

p12

early recent / established advanced

Spectrum Spectrum of anti-HIV of anti-HIV

testing testing

Hasil interpretasi :. Tidak ditemukan di pita2 (bands), negative. Supaya hasil dikatakan positif bila ada 3

bands yang melawan antigen yang harus didapatkan yaitu : p24, p31, gp41 atau gp120/160.

CDC criteria memerlukan 2 bands dari: p24, gp41 or gp120/160.

Mahal : $ 80 - 100 Teknik Lebih Sulit Interpretasi scr visual ‘Gold Standard’ untuk

Konfirmasi

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, Grave’s

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.

Dapat digunakan utk mendeteksi virus maupun antibodinya.

Antibody dideteksi dengan test serum pasien melawan antigen yang dipaparkan pada slide, diinkubasi, dicuci dan ditambah flourescent antibodi.

Virus dideteksi oleh sel pasien yang difiksasi dan diinkubasi dengan antibodi.

Pemeriksaan p24-antigen adalah metode EIA yg menggunakan bahan serum atau plasma.

P24 antigen hanya muncul pada waktu yg singkat, menghilang ketika terbentuk antibodi terhadap p24 muncul.

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.

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 tidak direkomendasi utk screening rutin sebab munculnya tidak bisa ditebak.

Sensitivitas lebih rendah dari ELISA.

Paling banyak digunakan utk:: Infeksi awal yang diduga pasien yg hasilnya

secara serologi negatif . Anak baru lahir deteksi cairan serebro spinalismonitoring progres penyakit

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 patient’s HIV DNA if present.

Radioactivity is determined.

Virus isolation dapat digunakan sebagai diagnosa HIV scr definitif.

Bahan terbaik adalah darah perifer tapi bisa menggunakan cairan sumsum, air liur, sekret cervik, semen dan airmata atau jaringan dari organ biopsi.

Pertumbuhan sel yg dibiakkan dirangsang dan jumlahnya dilipatgandakan utk menghasilkan virus.

Kultur diinkubasi selama 1 bulan, konfirmasi infeksi terdeteksi p24 antigen yg berada di supernatan.

Viral load atau viral burden adalah jumlah dari HIV-RNA yang berada dalam darah dengan menggunakan metode PCR.

RNA adalah material genetik dari HIV yang berisi informasi utk membuat lebih banyak virus.

Viral load tests diukur jumlah dari HIV dalam satu mililiter darah.

Perlu dua kali pengukuran dengan jarak 2-3 minggu utk menentukan jumlah rata2nya.

Diulang setiap 3-6 bulan dihubungkan dengan jumlah CD 4 utk memonitor viral load dan jumlah T-cell.

Diulang 4-6 minggu setelah memulai atau merubah obat anti retroviral utk menentukan efeknya terhadap viral load.

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.

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