Serology Test for Infectious Disesase: What & When · 06.03.2019 · Serology Test for Infectious...
Transcript of Serology Test for Infectious Disesase: What & When · 06.03.2019 · Serology Test for Infectious...
Serology Test for Infectious Disesase:What & When
Umi S. Intansari
Departemen Patologi Klinik & Kedokteran Laboratorium
Fakultas Kedokteran
Kesehatan Masyarakat & Keperawatan
PERTANYAAN SEPUTAR DIAGNOSTIK LAB
TERKAIT INFEKSI
Pemeriksaan apa yang bisa dilakukan? Parameter apa saja?
Kapan sebaiknya mulai diperiksa darah ?
Bagaimana interpretasi hasil lab serologi?
Apa pemeriksaan lab untuk diagnosis pasti ?
Bagaimana memantau perjalanan penyakit denganpemeriksaan lab ?
The choice of diagnostic method depends on:
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• clinical diagnosis
• epidemiological survey,
• Research, vaccine development
Purpose of the tests
laboratory facilities
technical expertise available
costs
the time of sample collection
Laboratory diagnostic method
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Ease of use
Confidence
DIRECT METHODS INDIRECT METHODS
Virus Isolation Genome
detection
Antigen
detection
Serology
IgM
Serology
IgG
TDR Cit. J. Cardosa
• The branch of laboratory medicine
• scientific study of blood serum
• In practice refers to the diagnostic identification of Ab (& Ag)
Serology
• Antigen and antibody reactions in vitro
• Detections and quantization of antigens and antibodies.
Serological test
Majority Diagnostic tests are serological test
Keith chaitoff, 2004 abbot diagnostic division
Antigen
+Antibody
+ Indicator system
/ detector
Immuno-serology techniqueWhat
Antigen antibody reactions (serological tests)
Precipitation reactions
Agglutination reactions
Virus neutralization test
Immuno fluorescence test
Radioimmuno assay
Enzyme- linked immunosorbent assay(ELISA
Immunoelectroblot eg., westren blot
Immunochromatographic tes
Flow cytometry, etc
Immunoassay
• Precipitation
• Agglutination
Unlabelled
immunoassay
• RIA, ELISA, CLIA, FIA
• Lateral flow Chromatography
• Flow cytometry
Labeled
immunoassay
Immuno-serological testing
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Figure A-10
Different Ab bind to distinct epitopes on Ag
Affinity?
Avidity?
Antigen-Antibody interaction
Antibody isotypes
IgM is the first to produced before isotypeswitching
After maturation B cell will express different set of isotypes depend on effector site:
low affinity but compensate by pentameric form
Figure A-9
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Precipitin curve
Agglutination
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Antibody can agglutinate to Ag on the surface of a large particle (bacteria, latex)
Hemaglutination if Ag on the surface RBC
• Widal test, C-Reactive Protein,
• ASTO, TPHA
Clinical application:
Agglutination
Haemagglutination Inhibition assay(Neutralization test)
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Neutralization
Test
Labelled Immunoassay
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Basic parameters:
• Solid phase 1 reactant
• Separation bound & free reagent
• Color development enzyme
Label:
• Enzyme: Enzyme-linked immunosorbent assay
• Chemiluminescense molc
• Radioactive molc
• Fluorescense molc
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Figure A-20
Flow cytometry
Wicking Material
Colloidal Gold Pad
• Flavivirus specific MAb
conjugated to gold colloid
• Dengue 1-4 Recombinant Antigens
Add 10µL of blood or serum
Membrane with Immobilised
Antibodies
• IgM capture line
• IgG capture line
• Control Line
Blood Separation Device
Absorbent Pad
Backing Sheet
IgM Capture Line
IgG Capture Line
Control Line
Add 2 drops of running
buffer
Rapid Assay
Cassette EnclosureRelease of Serum
Components
Release of Assay
Reagents
Antibody Complexing
15 min
Processing Center
Supply Center
Matrix Cell Hopper
System Control Center
Sampling
Center
Automation
Interpretation
• Qualitative:
• Positive/ Negative
• Optical density, Index value : ODs/Odst, Cutoff
• Quantitative: analyte consentrationunit/mL
Results
Method & performance
Course & history of disease
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Interpretation of Ag Detection
Recent infection
• HBsAg : acute/ chronic infection/ carrier
• HBeAg : replication marker, infectious
Depend on the marker:
Qualitative/ quantitative
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Interpretation of Ab Detection
•IgM : recent•IgG : recent/ past/secondary paired sera?•Total
Recent/ Past infection :
• Anti HBs: protective• Anti HCV & anti HIV: diagnostic
Depend on the marker:
• high • low
Avidity
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Innate versus Adaptive Mechanisms
When
• Seroconversion:
• development of detectable Ab in serum
Seroconversion
• Seroreversion:
• The opposite of seroconversion
• When test is no longer detect Ag/Abin patient’s sera
Seroreversion
Primary versus Secondary Response
IMMUNE RESPONSE
Primary Antibody response:
• First exposure to ag
• IgM appears 3-5 days,
increased and then drops
over a few weeks-months
• IgG detectable 1-2 weeks ,
increased and decreases
over a period of time
Secondary Antibody response/ Anamnestic response:
• After reexposure to ag
• Antibody production increases rapidly
• IgG increase in 2 – 3 days and
increases higher levels than primary
response and remains detectable for
months or years
Antibody Titer• Antibody titre is the
concentration of antibodies against a particular antigen
• Serology test is usually done using micro well plate.
• So that the test sample can be done in a very small sample.
Results Titers provide more valuable information when tested in parallel with a previous titer specimen.
A comparison of the current specimen’s results and previous specimen’s current results should be made.
A change in titer of 2 or more tubes is considered to be significant.
Early Diagnosis Late Diagnosis
days-4 0 4 6 14-21 >50
Hematology Testing
Molecular Detection (RT-PCR)
Virus Isolation
Viral Antigen Detection
Neutralisation Test
Hemaglutination Inhibition
Serology Methods IgG / IgM
By Elisa or Rapid Tests
Timing is critical in the Use of Dengue Diagnostics
Course of dengue infection and timings of diagnosis
Interpretation of Den Diagnostic tests
No single diagnostic assay can diagnose all dengue patients at the various times they may present with symptoms.
WHO, 2011
Interpretasi
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Respon
Antibodi
Interval
S1-S2
Titer S2 Interpretasi
4X 7 hr 1:1280 Infeksi akut, primer
4X Random 1:2560 Infeksi akut, sekunder
4X < 7 hr 1:1280 Infeksi akut, primer/sekunder
tetap Apapun >1:2560 Infeksi sekunder, baru terjadi
tetap 7 hr 1:1280 Bukan dengue
tetap Spesimen
tunggal
1:1280 Tak dpt diinterpretasi
HIV Time Line
Acute Hepatitis B Virus Infection with Recovery
Typical Serologic Course
IgM anti-HBc
HBsAg
0 4 8 12 16 20 24 28 32 36 52 100
Weeks after Exposure
Titer
HBeAg anti-HBe
Symptoms
anti-HBs
Total anti-HBc
HBV DNA
Window
Period
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Primary Infection
IgM: day 5 after onset rise for 1-3 weeks, up to 60 days.
IgG: day 10-14 after onset of fever and persists for life.
Secondary Infection
• IgM: Produced at low or undetectable levels
• IgG: Rising rapidly 1-2 days after onset of symptoms
I had high fever last night.
So I go to a hospital this morning.Dr. said it is not dengue fever.
Because they tested Dengue IgG/IgM.
Case 1 *for example
SymptomNS1 Ag
DAY -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 12
AntibodyBite
Ag/A
b level
Day
NS1 Ag
IgM
IgG
Case 2 *for example
DAY -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 12
AntibodyBite
I had high fever from 1 week ago.
So I just took a aspirin.
But I could not get over, so I visit
hospital this morning.
Dr. said it is not dengue fever.
Because they tested Dengue NS1 Ag.
SymptomNS1 Ag
Ag/A
ble
vel
Day
NS1 Ag
IgM
IgG
1 2 3 5 7 10 12