ASPEK LABORATORIUM HIV-AIDS

Neshya R.P
Tutor : Dr. dr. Tinny SpPK(K)
 Pendahuluan
 Defisiensi sistem imun sering terjadi bukan
akibat genetik, melainkan karena didapat
selama hidup, salah satunya akibat terinfeksi
virus HIV.
 AIDS (Acquired Immunodeficiency Syndrome):
penyakit akibat infeksi HIV (Human
Immunodeficiency Virus)
Ditandai dg imunosupresi yg berhubungan dg infeksi
opportunistik dan malignant tumors, wasting, dan
degenerasi central nervous system (CNS).
 HIV menginfeksi sel-sel imun : CD4-expressing
helper T cells, macrophages, dan dendritic
cells.
Karakteristik HIV
 HIV : anggota famili lentivirus dari retrovirus
binatang.
 Lentivirus mempunyai kemampuan menimbulkan
infeksi laten, cytopathic effects, dan yg lebih fatal
yakni wasting syndromes dan degenerasi CNS.
 Ada 2 tipe HIV :
HIV-1  terbanyak, penyebab utama AIDS
HIV-2  >> di Afrika, tidak seganas HIV-1
 Structure of HIV-1

Core :
 2 strain identical RNA (the viral
genome)
 Enzymes : reverse transcriptase,
integrase, and protease
 p24 capsid protein with a surrounding
p17 protein matrix
Envelope :
 phospholipid membrane envelope
derived from the host cell.
 gp41 and gp120 are bound to the
envelope  These subunits are
produced by proteolytic cleavage of a
gp160 precursor.

CD4- and chemokine receptors on the

host cell surface  HIV-1 receptors
 Perjalanan Infeksi HIV
 Segera setelah virus
masuk ke aliran darah, HIV
replikasi cepat, dan viral
load meingkat tajam
 Banyak sel CD4
dihancurkan  jumlah
turun drastis.
 Setelah bbrp minggu,
sistem imun mulai
membentuk antibodi thd
HIV  viral load mulai
menurun dan jumlah CD4
meningkat kembali.
 Antibodi baru dapat
terdeteksi setelah
beberapa minggu. Pada
masa ini, viral load dan
daya menular paling tinggi.
 Kelanjutan infeksi setelah
infeksi akut.
 Tahap ini dimulai dengan
masa tanpa gejala, yg
bertahan rata-rata 7-10
tahun/lebih/kurang.
 Selama masa ini viral load
meningkat pelan-pelan,
sementara jumlah CD4
terus-menerus merosot
 HIV replikasi terus
kemudian viral load mulai
meningkat tajam,
sementara jumlah CD4
menurun < 200/mm3 
didefinisikan AIDS.
 PEMERIKSAAN LABORATORIUM
HIV
 Deteksi Antibodi thd protein envelope atau Core HIV :
ELISA
Recombigen Latex Agglutination Test
Western Blot
Indirect Immunofluorescence Assay (IFA)
Radio Immunoprecipitation Assay (RIPA)  utk penelitian
Rapid test  Immunochromatography (ICT)
 Identifikasi antigen HIV :
 ELISA  gp 24
 PCR  viral load
ELISA
- Detect antibodies to HIV-1 and HIV-2.
- HIV Ag / Ab reaction is detected by color
change
- Intensity of color reflects amount of
antibody present serum
ELISA…
 Interpretasi hasil tes :
Sampel (+) mengandung anti-HIV bila memberi densitas
optis < nilai cut-off absorbance
Semua serum yg (+) diuji ulang dg uji konfirmatif Western
Blot.
 Kelemahan tes :
Dapat memberikan hasil (+) / (-) semu, shg harus
dikonfirmasi dg Western Blot.
Pada fase awal infeksi, terutama saat Window period, uji
ELISA anti-HIV dapat (-) semu  shg pada awal infeksi
sebaiknya dipakai uji ELISA Ag HIV atau uji PCR.
Serum lipemik, ikterik atau terkontaminasi  (+) semu
 Karakteristik tes :
Kecuali pada window period, sensitifitas uji ELISA anti-HIV
hampir 100%, spesifitas 99,3-99,9%.
ELISA : HIV p24 Antigen

 HIV p24 : Core protein of the virus

 Detects p24 antigen before antibody can be
detected
 Detected 2 - 3 weeks after HIV infection
 Detected about 6 days before antibody tests
become reactive
 Used for:
Diagnosis of pediatric HIV-1 infections
Blood bank safety (high incidence countries)
 Issues:
Level 4 complexity
Properly maintained equipment required
Western Blot Immunoassays
 Used as supplemental test for
confirmation (only difficult cases)
 Detects antibodies to specific HIV
antigens on cellulose strip
 Issues:
Multiple standards for performance and
interpretation
Expensive
Limited commercial availability
 Cara kerja tes Western Blot

Ag HIV murni pada polyacrylamide gel

Separasi dg elektroforesis

dipindahkan ke nitrocellulose.

Nitrocellulose diinkubasikan dengan serum penderita.

Antibodi HIV dideteksi dengan memberikan antlbodi anti-human yg sudah

dikonjugasi dg enzim  menghasilkan wama bila diberi substrat.

Gambaran band dari bermacam-macam protein envelope dan

core dapat mengidentifikasi macam antigen HIV.
Western blotting..
 Interpretasi :
(+) : bila pita yg terdeteksi meliputi p24, p31, dan gp41 atau
gp120/160
(-) : tidak terlihat pita HIV spesifik,
pola yg lain disebut indeterminate.
Menurut US CDC (Centers for Disease Control) :
• (+) : terdeteksi 2 pita dari p24, gp41 dan gp120/160
• Intermediate : satu pita saja yg terdeteksi  p24, gp41,
gp120/160, p66, p55, p51, p31 atau p17
• (-) : tidak ada pita yg terdeteksi
 Karakteristik tes :
Sensitifitas dan Spesifitas tinggi  dipakai utk tes konfirmasi
HIV (+) dg ELISA
Latex Agglutination Test
Prinsip dasar :
Antigen-coated latex particles (gp 41 and gp 120) + Anti-HIV
antibodies (serum Px)  agglutination.

Antigen
HIV

Antibody
(serum Px)
 Latex Agglutination Test…
 Interpretasi hasil tes :
Rentang hasil : 1+ sampai 4+
(+) : gumpalan amat halus dg latar
belakang putih susu
(++++) : gumpalan partikel yg lebih
besar tanpa latar belakang putih susu
(-) : aglutinasi (-), warna putih susu
 Karakteristik tes : (+) (-)
Sensitifitas dan spesifitas < ELISA
Sensitifitas 99,4%, spesifitas 99,5%.
 Indirect Immunofluorescence Assay (IFA)

 Indirect
First Ab to Ag is unlabeled
Fluorochrome
Fluorochrome-labeled Labeled Anti-Ig
anti-Ig is used to detect Unlabeled
Ab
binding of the first Ab.

Ag
HIV Rapid Tests
 Qualitative assay to detect HIV antibodies
 Most detect HIV 1 and HIV 2
 Body Fluids Used for HIV Rapid Testing :
Serum
Plasma
Whole blood
Oral fluids
 Three Formats of HIV Rapid Tests :
Immunoconcentration (flow-through device)
Immunochromatography (lateral flow)
Particle agglutination
HIV Rapid Tests
Advantages Disadvantages
1. Supports increased number 1. Small numbers for each test
of testing sites run
2. Same-day diagnosis and 2. Quality Assurance/Quality
counseling Control at multiple sites
3. Easy to use 3. Test performance varies by
4. Test time under 30 minutes product
5. None or one reagent 4. Refrigeration required by
6. Minimal or no equipment some products, e.g., Capillus
required 5. Reader variability in
7. Minimum technical skill interpretation of results
8. Increases access to 6. Limited end-point stability of
prevention (VCT) and test results
interventions (PMTCT)
CD4 T-Lymphocyte
 CD4 T-lymphocyte counts used
for:
 Determining clinical prognosis
 Assessing criteria for antiretroviral
therapy
 Monitoring therapy
 Flow cytometry is the standard
method for the estimation of
CD4/CD8 counts
 Issues:
 Requires high level of technical skill
for test performance and interpretation
 Properly maintained equipment
Viral Load
• Quantitative molecular assay measures amount of HIV in
blood
• Used to:
– Predict disease progression
– Assist with deciding when to initiate anti-retroviral therapy
– Monitors response to anti-retrovirals
• Issues:
– Expensive
– Labor-intensive
– Special facilities

The HIV-1 specimen is plasma collected in EDTA that must be

separated from the cells within 6 hours.
Heparin cannot be used as an anticoagulant because it
inhibits PCR.
PCR
Deteksi virus secara langsung dapat
dipertimbangkan sebab :
1. Infeksi virus HIV yg laten  uji langsung dapat
membantu identifikasi individu yg terinfeksi virus
tanpa adanya perubahan serologis.
2. Mendeteksi HIV pada bayi  zat anti maternal
dapat bertahan sampai 15 bulan shg sulit untuk
dibedakan dengan zat anti bayi.
3. Membantu menentukan status individu dengan
hasil Western Blot negatif ke dalam kelompok
risiko tinggi atau rendah.
PCR…
• Pemeriksaan ini paling baik digunakan untuk :
– tersangka penderita, yang secara serologi tidak
jelas menunjukkan adanya infeksi, termasuk anak-
anak dari ibu seropositif
– pasangan yang salah satunya seropositif;
– pemakai obat secara intravena
– individu yang meskipun tidak secara pasti
seropositif tetapi mungkin terkena atau tertular
infeksi dari retrovirus yang lain
– dapat untuk memeriksa darah dari sejumlah besar
donor untuk memastikan apakah mereka bebas
virus
TERIMA KASIH
wasting syndrome
 = a condition characterized by weight loss
associated with chronic fever and diarrhea.
 Over a period of 1 month, the patient may
lose 10% of baseline body weight.
 In cases of human immunodeficiency virus
infection, the malnutrition of wasting
exacerbates the condition.
 Several factors contribute to AIDS wasting:
 Low food intake:
 Low appetite is common with HIV.
 Some AIDS drugs have to be taken with an empty stomach, or with a
meal.
 Drug side effects such as nausea, changes in the sense of taste, or
tingling around the mouth also decrease appetite.
 Opportunistic infections in the mouth or throat can make it painful to
eat. Infections in the gut can make people feel full after eating just a
little food. Finally, lack of money or energy may make it difficult to
shop for food or prepare meals.
 Poor nutrient absorption:
 Healthy people absorb nutrients through the small intestine.
 In HIV disease, several infections (including parasites) can interfere
with this process.
 HIV may directly affect the intestinal lining and reduce nutrient
absorption. Diarrhea causes loss of calories and nutrients.
 Altered metabolism:
 Food processing and protein building are affected by HIV disease.
Even before any symptoms show up, you need more energy. This
might be caused by the increased activity of the immune system.
People with HIV need more calories just to maintain their body
weight.
A Reverse transcriptase enzyme
 = RNA-dependent DNA polymerase
 is a DNA polymerase enzyme that transcribes
single-stranded RNA into double-stranded DNA.
 It also helps in the formation of a double helix DNA
once the RNA has been reverse transcribed into a
single strand cDNA.
 Reverse-transcribing RNA viruses, such as
retroviruses, use the enzyme to reverse-transcribe
their RNA genomes into DNA, which is then
integrated into the host genome and replicated
along with it.
 Clinical latency

 Clinical latency is the state, or time period, in which

an infectious agent such as a virus or bacterium
replicates in its host without causing medical signs in
the host.
 With respect to viral infections, in clinical latency the
virus is actively replicating. This is in contrast to viral
latency, a form of dormancy in which the virus does
not replicate.
 During clinical latency, an infection is subclinical.
 HIV Window Period
 HIV window period is the time between the contraction of
the disease and production of the antibodies.
 It can be named as the first or the initial stage of HIV
infection.
 HIV test, conducted during the window period cannot detect
any traces of HIV antibodies and the result of such antibody
tests, like ELISA, Rapid Test or the Western Blot, would be
fallaciously negative.
 On an average, the window period lasts for 2 to 12 weeks.
 A HIV test proves positive only after the termination of the
HIV window period when enough anti bodies have been
produced within our system.
 HIV Window Period States:
 Even though HIV virus are present in the blood, there would
be no HIV anti bodies in the blood
 As the tests cannot detect the presence of these antibodies,
the result will be 'HIV Negative'
 The patient during this period is capable of infecting others.
 Complexity of HIV Tests Varies*

• Level 1: No additional equipment and little or no

laboratory experience needed
• Level 2: Reagent preparation or a multi-step process
is required; Centrifugation or optimal equipment
• Level 3: Specific skills such as diluting are required
• Level 4: Equipment and trained laboratory technician
are required

*WHO Reports

Lab workers Health workers

PCR
 PCR : cara in vitro untuk memperbanyak target
sekuen spesifik antigen untuk analisis cepat atau
karakterisasi, walaupun material yang digunakan
pada awal pemeriksaan sangat sedikit.
 Pada dasarnya PCR meliputi tiga perlakuan yaitu :
Denaturisasi
hibridisasi dari "primer" sekuens DNA pada
bagian tertentu yang diinginkan
perbanyakan bagian tersebut oleh Tag
polymerase
 Dikerjakan dg mengadakan campuran reaksi
dalam tabung mikro yang kemudian diletakkan
pada blok pemanas yang telah diprogram pada
seri temperatur yang diinginkan.
HIV-1 genome
Mechanism of HIV entry into a cell
HIV life cycle
Steps in HIV Infection and Pathogenesis
 Respon Immun terhadap Infeksi HIV

 Terdapat respon humoral dan cell-mediated immune responses

specific pada pasien yg terinfeksi HIV.
 CTL respons thd HIV terdeteksi 2-3 minggu setelah infeksi
awal  puncak : minggu 9-12, dilanjutkan ekspansi yg nyata
dari klon virus-specific CD8+ T cell .
 Respon imun humoral mencapai puncaknya pada minggu ke-
12.
 Mechanisms of Immune Evasion by HIV
 HIV has an extremely high mutation rate because of
error-prone reverse transcription, and in this way it may evade
detection by antibodies or T cells generated in response to viral
proteins.
 HIV-infected cells may evade CTLs through down-
regulation of class I MHC molecule expression,
particularly HLA-A and HLA-B proteins, mainly by promoting
internalization of these molecules.
 HIV infection may inhibit cell-mediated immunity.
In infected individuals, TH2 cells specific for HIV and other
microbes may expand relative to TH1 cells.
Because TH2 cytokines inhibit cell-mediated immunity  this
imbalance forms dysregulation (immune deviation) that
increases host susceptibility to infection by intracellular
microbes, including HIV itself.
Mechanisms of Immunodeficiency
 HIV infection ultimately results in impaired
function of both the adaptive and innate
immune systems.
 The most prominent defects are in cell-
mediated immunity
 An important cause of the loss of CD4+ T
cells in HIV-infected people is the direct
cytopathic effects of infection of these cells
by HIV
Several direct toxic effects of HIV on infected CD4+ cells
 The process of virus production, with expression of gp41
in the plasma membrane and budding of viral particles,
may lead to increased plasma membrane permeability
and the influx of lethal amounts of calcium, which
induces apoptosis, or osmotic lysis of the cell caused by
the influx of water.
 Viral production can interfere with cellular protein
synthesis and expression and thereby lead to cell death.
 Unintegrated viral DNA in the cytoplasm of infected
cells, or large amounts of nonfunctional viral RNA, may
be toxic to the infected cells.
 The plasma membranes of HIV-infected T cells fuse with
uninfected CD4+ T cells by virtue of gp120-CD4
interactions, and multinucleated giant cells or syncytia
are formed  can be lethal to HIV-infected T cells as
well as to uninfected CD4+ T cells that fuse to the
infected cells.
Clinical Features of HIV Infection
Tests Based on Immunoconcentration

Flow-Through Devices:
Multi-Spot
Genie II
Top view

Reading Results: Genie II

Side view

Non-reactive Reactive
How Immunoconcentration Works
HIV antibody (serum Px) links to bound HIV
peptide antigens forming the color spot

Internal Control
HIV-1 peptide

HIV-2 peptide
How Immunochromatography Works

Add
Sample Test Control
Conjugate
Line line

IgG Antibodies Colloidal gold HIV antigen Anti-IgG/gold

HIV antibodies conjugated to antibodies
HIV antigen
Tests Based on Immunochromatography

Lateral Flow Devices

Determine
Hema-Strip Control

OraQuick HIV Antigen

Unigold

Sample pad
Specimen Flow
Reading Results: Determine

Non-
Reactive
Reactive

Sample Pad Test line Control line

Applications of PCR
A 142 base target sequence in the HIV-1 gag gene
is converted from RNA to complementary DNA,
and to double stranded DNA using Thermus
thermophilus DNA polymerase in the presence
of manganese and buffers, which performs the
reverse transcription and the amplification steps
simultaneously.
The standard specimen procedure can quantitate
HIV-1 RNA in a range of 400-75,000 copies/mL.