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 HIV2 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 T4lymphocytes.

(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 doublestranded 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 mothers CD 4 count.

 spt

infeksi Mononucleosis-like, gejala spt batuk pilek dapat timbul 6 -12 weeks setelah infeksi :
lymphadenopathy Demam Rash ( bercak kemerahan di tubuh ) Pusing Lelah DIare Sakit tenggorokam Manifestasi 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 leukoplakia Kaposis 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

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.

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

Mycobacterium avium Cytomegalovirus infections lymphoma dementia Banyak terjadi kematian padaCD4 < 50.

Bila dihitung CD4 < 50:

Respiratory system

Pneumocystis Carinii Pneumonia (PCP) Tuberculosis (TB) Kaposi's Sarcoma (KS) Cryptosporidiosis Candida Cytomegolavirus (CMV) Isosporiasis Kaposi's Sarcoma Cytomegolavirus Toxoplasmosis Cryptococcosis Non Hodgkin's lymphoma Varicella Zoster Herpes simplex Herpes simple Kaposi's sarcoma Varicella Zoster

Gastro-intestinal system

Central/peripheral Nervous system

Skin

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 gp41 As 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 Tcell 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 Burdens

Regimen 1996
Zerit/Epivir/Crixivan 10 pills, Q8H

Dosing

Daily pill burden

1998
Retrovir/Epivir/Sustiva 5 pills, BID

2002
Combivir (AZT/3TC)/EFV 3 pills, BID

2003
Viread/ Emtriva/Sustiva

3 pills, QD 2 pills, QD

2004
Truvada/Sustiva

symptoms

symptoms

HIV proviral DNA HIV antibodies window period

HIV viral load

HIV-1 p24 antigen

0 1 1 infection 2 3
weeks

6
years

10

Time following infection

 Menggunakan

metode yg mendeteksi:

Antibody Antigen Viral 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. 1. Sensitivitas reagen pertama >99% 2. Spesifisitas reagen kedua > 98% 3. Spesifitas reagen ketiga > 95% reagen pertama harus memiliki sensitiviras >99%. Spesifisitas reagen kedua >98%. Semua regensia yang dipakai harus terdaftar di DepKes RI

Strategi III dengan persyaratan reagensia sebagai berikut :

Untuk tujuan surveilans :

 Pemeriksaan

anti-HIV disertai informed consent tertulis, sebelumnya didahului dengan konseling prauji / 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 infeksi Penelitian.

 Ada

3 teknik metode yang digunakan:

indirect competitive sandwich

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.

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,

Test Konfirmasi yang paling populer :

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.

gp160 gp120

p68 p55 p53 gp41-45

Spectrum of anti-HIV testing

early
DNA PCR RNA PCR p24 Ag 3rd gen ELISA 1st gen ELISA Detuned ELISA

p40 p34 p24 p18 p12

recent / established advanced

1wk

2wk

3wk

2mo 6mo

1yr 2yr 3yr

+8yr

 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, 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.

 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 spinalis monitoring 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 patients 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 23 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.h tml#translat

http://pathmicro.med.sc.edu/lecture/HI

V3.htm
http://www.avert.org/hivstages.htm
http://www.aidsinfo.nih.gov/guidelines/

http://www.hopkinsaids.edu/publications/pocketguide/pocketgd0105.pdf

http://www.modares.ac.ir/sci/saman_h/Pages/application s.htm http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-0202-02-02 http://www.hivandhepatitis.com/recent/test/realtime/06 1604_f.html