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NATANIEL TANDIROGANG

LABORATORIUM MIKROBIOLOGI
FK UNMUL
2014
Imunitas
Reaksi tubuh thd masuknya substansi asing
Respon imun
Kumpulan respon thd substansi asing yg
terkoordinasi
Sistem imun
Sel & molekul yg bertanggung jawad dlm
imunitas
Melindungi tubuh dengan mengenali antigen pada
bakteri/ virus
Terdiri dari dari Organ lymphoid
Semua komponen penting untuk produksi dan
pematangan limfosit
Sel T dan Sel B diproduksi oleh stem sel di sumsum
tulang dan jaringan limfoid
Sel B
mengenali antigen spesifik dan menghasilkan antibodi
spesifik
Antibodi bekerja dengan membungkus antigen lalu
memicu sistim komplemen
Membungkus antigen & membuat antigen rentan thd
fagosit
Ada 5 Kelas :G,A,M,D,E
Sel T
Ada 2 fungsi: regulasi sistem imun dan membunuh sel-sel yang
membawa target antigen spesifik.
Setiap sel T memiliki penanda permukaan, seperti CD3, CD4, CD8 yg
membedakan antar sel
CD4+ merupakan sel pembantu yang mengaktivasi sel B, killer cells,
dan makrofag saat ada antigen spesifik.
CD8+ membunuh sel yang terinfeksi virus atau bakteri, juga sel-sel
kanker
mampu menghasilkan sitokin (zat kimia yang dapat membunuh sel)
seperti interferon
Sitokin juga meningkatkan perumbuhan sel, mengaktivasi fagosit dan
menghancurkan sel target.
Interleukin merupakan jenis sitokin yang berperan sebagai pembawa
pesan antar sel darah putih
Fagosit
Fagosit terdiri atas monosit dan makrofag
Fungsi :menelan dan mencerna sel yang membawa
partikel antigen.
memulai respon imun dengan mempresentasikan antigen
kepada limfosit, dan penting sebagai regulasi respon imun
dan inflamasi
Sel dendritik, tipe lain dari fagosit, juga termasuk antigen-
presenting cells
Neutrofil adalah fagosit granulosistik yang penting dalam
respon inflamasi.
Fig. Action of Cytokines in networking
- Tuberculosis (phthisis) described since the
time of Hippocrates (460 BC - 370 BC)

- 1689: Doctor Richard Morton used the


term consumption to denote TB.

- Second half of the 17th century: high


death rates from TB in Europe.

- 1722: Doctor Benjamin Marten proposed


that TB could be transmitted in the air and
described TB as being caused by
wonderfully minute living creatures

- End of 19th century to the start of 20th


century: Principal cause of death in
Europe was TB.
- The romantic Era of TB
Queen Guinevere painted by William Morris
- 1865 Jean-Antoine Villemin: confirmed that
TB is contagious.

- Robert Koch:
- 1882: Isolated and cultured M.
tuberculosis.
- 1890: Announced the discovery of
tuberculin.
- Developed staining methods used to
identify the bacteria.
- 1905: Received the Nobel Prize

- Bacteriologist Paul Ehrlich developed Ziehl-


Neelsen staining.
Visualization of M. tuberculosis
using the Ziehl-Neelsen stain
- Late 1800s: Edward Livingston Trudeau
established Adirondack Cottage
Sanatorium, first TB sanatorium in the US.
- 1896 Theobald Smith demonstrated that
bovine TB is caused by M. bovis.
- 1908 Albert Calmette and Camille Gurin
isolated M. bovis and grew it in ox bile.
- Identified a morphological variant of M.
bovis found to be avirulent, conferred
immunity against M. tuberculosis.
Lead to the BCG vaccine (bacilli Calmette-
Gurin).
- Development of antibiotics to combat
infection:
1947: streptomycin, 1952: isoniazid M. bovis

The majority of drugs used to combat


infection were identified between 1945 and
1967.
No new drugs developed since the 1980s
- Reoccurrence of TB for two main reasons:
1)HIV/AIDS pandemic
2)Development of drug resistance
General Characteristics
- Family Myobacteria
- Gram-positive aerobic rod-shaped bacilli
- Acid fast bacteria
- Lack of spore formation and toxin production
- No capsule, flagellum (non-motile)
- Generation time of 18- 24 hours but requires 3-4
SEM of M. tuberculosis
weeks for visual colonies
Pathological Features
- Principle cause of Human Tuberculosis
- Intracellular pathogen (alveolar macrophages)
- Waxy, thick, complex cellular envelope
- Cell envelope components ex) sulfolipids
- Produces tubercles, localized lesions of M.
tuberculosis
M. Tuberculosis
(stained in purple)
General Features
- Thick, waxy and complex
- Higher fluidity in more external
regions than internal regions
- Relatively impermeable to
hydrophilic solutes
- Contain porins (selective cationic
channels)
Main Components
- Peptidoglycan
contains N-glycolylmuramic
acid instead of N-acetylmuramic
acid
- Arabinogalactan
- Mycolic Acids (60% of cellular
envelope)
- Lipoarabinomannan (LAM)
Resistance to Drying and Other Environmental Factors
- Thick, waxy nature of cellular envelope protects M. tuberculosis
from drying, alkali conditions, and chemical disinfectants
- Hinders entrance of antimicrobial agents
Entry into Host Cells
- Lipoarabinomannan (LAM) binds to mannose
receptors on alveolar macropages leading to
entry into the cell

Interference of Host Immune Response


- Glycolipids and sulfolipids decrease the effects of oxidative
cytotoxic mechanism
- Inhibition of phagosome and lysosome fusion inside macrophage
- Waxy cellular envelope prevents acidification of the bacteria inside
the phagosome
Dual roles during TB
infection
Preferred host cell
Effector cell

Effector cell function is


cell-mediated
Requires cytokine
signal from CD4+ T
helper cell
Macrophages must first
present antigen bound
to MHC class II
tahun 1983 , Jean Claude Chermann & Francoise Barre-
Sinoussi dari Perancis ALV ( Lymphadenopathy- Associated
Virus)

Bersama dengan Luc Montagneir, mereka membuktikan


bahwa virus tersebut merupakan penyebab AIDS

Awal tahun 1984, Robert Gallo dari Amerika Serikat


HTVL- III (Human T-cell Leukemia virus III ) . Setelah
diteliti lebih lanjut, terbukti bahwa ALV dan HTLV-III
merupakan virus yang sama

Tahun 1986, istilah yang digunakan untuk menyebut virus


tersebut adalah HIV, atau lebih spesifik lagi disebut HIV- 1.
Tidak lama setelah HIV-1 ditemukan, suatu subtipe baru
ditemukan di Portugal dari pasien yang berasal dari Afrika Barat,
dan kemudian disebut HIV-2.

Melalui kloning dan analisis sekuens ( susunan genetik ), HIV-2


memiliki perbedaan sebesar 55 % dari HIV-1 dan secara antigenic
berbeda. Perbedaan terbesar lainnya antara kedua strain (galur)
virus tersebut terletak pada glikoprotein selubung.

Penelitian lanjutan memperkirakan bahwa HIV-2 berasal dari SIV


(Retrovirus yang menginfeksi primata ) karena adanya kemiripan
sekuens dan reaksi silang antara antibody terhadap kedua jenis
virus tersebut.
Etiologic agent of Acquired Immunodeficiency
Syndrome (AIDS).
Discovered independently by Luc Montagnier of
France and Robert Gallo of the 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)
Source: WHO/UNAIDS/UN The Millennium Development Goals Report, 2009, p.32 and WHO.
Ditjen PP & PL Kemenkes RI

New cases (Oct- Des 2012)


HIV: 6139 ; AIDS: 2145
HIV & AIDS have reported during 1 Januari -31
Desember 2012
HIV: 21511 ; AIDS: 5686
HIV & AIDS have reported during 1 Januari 1987 - 31
Desember 2012
HIV: 98390 ; AIDS: 45499
International Statistical Classification of Disease
and Related Health Problems Codes :

Kelas VI ( Virus SsRNA RT )

Famili Retroviridae

Genus Lentivirus

Spesies - Human Immunodeficiency


Virus 1
- Human Immunodeficiency
Virus 2
Icosahedral (20 sided), enveloped virus of the
lentivirus subfamily of retroviruses.
Retroviruses transcribe RNA to DNA.
Two viral strands of RNA
found in core surrounded by
protein outer coat.
Outer envelope contains a lipid
matrix within which specific
viral glycoproteins are
imbedded.
These knob-like structures
responsible for binding to
target cell.
Envelop
gp 120
gp41
Enzym
Reverse transcriptase
Integrase
Protease

Inti
P17 (matrix)
P24 (kapsid)
P7/P9 (nucleocapsid)
41
Perlengketan
Penetrasi
Limfosit T

45

45
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.
CD 4 KEMATIAN

Infeksi
Oportunistik
Viral
load

PERIODE
JENDELA Tanpa Gejala Gejala Klinis

3 Bulan 1 th 5 th 7 th 10-11 th 49

49
Tuberculosis
bacteria

Immune cell
Macrophage
I have TB!
TNF-
Immune cell
CD 4 Cell
Cells talk!
Antigen-presentation
CD 4 cells
activated!
Mobilise against Mobilise against
the TB infection! the TB infection!
IFN-
IFN-
HIV
Kandidiasis (jamur) di mulut, kerongkongan, vagina
Sitomegalovirus kebutaan
Herpes simpleks di mulut, kelamin
Mycobacterium Avium Complex (MAC) demam
kambuhan, rasa sakit menyeluruh, gangguan cerna,
BB turun drastis.
Pneumonia Pneumocystis Carinii (PCP)
Toksoplasmosis : infeksi protozoa otak
Tuberkulosis Paru Meningitis TB

64
65
66
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
68
69
70
71
72
73
74
75
76
77
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.
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
Methods utilized to detect:
Antibody
Antigen
Viral nucleic acid
Virus in culture
First serological test developed to detect HIV
infection.
Easy to perform.
Easily adapted to batch testing.
Highly sensitive and specific.
Antibodies detected in ELISA include those directed
against: p24, gp120, gp160 and gp41, detected first
in infection and appear in most individuals
ELISA tests useful for:
Screening blood products.
Diagnosing and monitoring patients.
Determining prevalence of infection.
Research investigations.
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.
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.
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.
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
Expensive $ 80 - 100
technically more difficult
visual interpretation
lack standardisation
- performance
- interpretation
- indeterminate reactions
resolution of ??
Gold Standard for
confirmation
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.
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.
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.
Most useful for the following:
early infection suspected in seronegative patient
newborns
CSF
monitoring disease progress
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 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 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 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.
Hingga 2005, jumlah ODHA di seluruh dunia
sebesar 40,3 juta orang dan yang terinfeksi HIV
sebesar 4,9 juta orang.
Pada 2008, 64 % dari seluruh kasus HIV terjadi di
Afrika dan 15 % terjadi di Asia Tenggara.
Di Indonesia (2009) 18442 kasus di 32 Provinsi.
Koinfeksi TB-HIV: 24% - 45% kasus TB pada
infeksi HIV asimptomatik dan sebanyak 70 % pada
pasien dengan AIDS
Kemungkinan 20-37 kali lipat
400.000 dari 1,7 juta orang yang
meninggal dari TB adalah TB-HIV
1,2 juta dari 9,4 juta kasus TB baru
adalah TB-HIV
Prevalensi TB-HIV berkorelasi dengan
prevalensi HIV
1. Mengetahui adanya Kecenderungan strain M.TB
menginfeksi Suku tertentu Kalimantan Timur
2. Mengetahui Galur infeksi M.TB di Kalimantan
Timur
3. Mengetahui Presentase IFN-Gamma Pada tiap Suku
di Kalimantan Timur
4. Mengetahui Hubungan keberhasilan Pengobatan
dengan Presentase IFN-Gamma