laboratory diagnosis of bacterial infection
TRANSCRIPT
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Laboratory Diagnosis ofBacterial Infection
Osman Sianipar
Department of Clinical PathologyGadjah Mada University, Yogyakarta
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Clinical Pathology
Clinical Chemistry
Hematology Serology
Microbiology
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Lab Diagnosis
Culture and Identification
Antigen detection Antibody detection
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Introduction
Microbiologist have relied on culture isolation of
microorganism to establish the etiology of bacterialinfection.
Isolation in pure culture and biochemical and/or
serologic identification of viable is still gold standard. Isolation is necessary if standard antimicrobial
susceptibility testing is to be performed on the
organism.
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Introduction
Some limitations to classic approach of cultureisolation and identification: Agent can not be cultivated on artificial media
Need cell culture
Labile in transport condition
Require a long incubation period
Agent may be fastidious
Give rise to difficulty to make early patient caredecision on the basis of culture results.
Some effort have been done in order to solvethese limitations.
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Culture and Identification
Source of specimen
Transport media
Culture technique: Automatic/manual
Media
Identification: Colony morphology
Staining, microscopic examination
Biochemistry Serologic technique
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Antigen detectionAntigen detection
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Antigen detection methods
Particle agglutination:
Latex agglutination
Staphylococcal co-agglutination Liposome-mediated agglutination
Precipitin tests:
Tube and agar precipitin
Counter-immuno-electrophoresis
Microscope-assisted labeled-antibody staining: Fluorescence-labeled antibody
Enzyme-labeled antibody
Particle agglutination:
Latex agglutination
Staphylococcal co-agglutination
Liposome-mediated agglutination
Precipitin tests: Tube and agar precipitin
Counter-immuno-electrophoresis
Microscope-assisted labeled-antibody staining: Fluorescence-labeled antibody
Enzyme-labeled antibody
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Antigen detection methods
Solid-phase immunoassay with labeled
reagent:
Enzyme immunoassay
Fluorescence immunoassay
Radioimmunoassay
Solid-phase immunoassay with labeled
reagent:
Enzyme immunoassay
Fluorescence immunoassay
Radioimmunoassay
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Particle agglutination
YY
YY +
Patient serum
(antigen)
YY
YY
Agglutination
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Antibody structure
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Particle agglutination
Antibody (IgG or IgM; polyclonal or monoclonal)
Reaction depends on:
Particle size
Avidity of antibody
Antibody type (polyclonal/monoclonal)
pH and ionic strength of the test specimen
Reaction temperature Antigen concentration
Antibody (IgG or IgM; polyclonal or monoclonal)
Reaction depends on:
Particle size
Avidity of antibody
Antibody type (polyclonal/monoclonal) pH and ionic strength of the test specimen
Reaction temperature
Antigen concentration
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Particle agglutination
Particle can be: Latex
Formalin-killedS. aureus
(protein A) Liposome
Particle can be: Latex
Formalin-killed S. aureus (protein A)
Liposome
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Latex agglutination
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Staphylococcal Coagglutination
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Liposome-mediated aglutination
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Tube and agar precipitin
(Precipitin tests)
Antigen
Antibody
AG well AB well
Precipitin band
Agar
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Counterimmunoelctrophoresis
(Precipitin tests)
AG well AB well
Agar coatedGlass slide
AGmigration
ABmigration
Buffer chamber
Cathode(-)
Anode(+)
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Fluorescence-labeled antibody
F
F
IndirectDirect
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Fluorescence-labeled antibody
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Enzyme-labeled antibody
E
E
IndirectDirect
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Enzyme-labeled antibody
Not require a fluorescent microscope
Improve sensitivity
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Solid-phase immunoassay with
labeled reagent More amenable to automation
Includes: Enzyme immunoassay
Fluorescence immunoassay
Radioimmunoassay
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EIA double AB
Conc
OD
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EIA, indirect double AB
Conc
OD
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EIA, competitive
Conc
OD
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M mbr n b nd lid ph EIA
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Membrane-bound solid-phase EIA
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Hybridization technique
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PCR
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PCR
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Van der Velden. Leukemia 2003 (www)
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Antibody detection
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Serologic diagnosis of bacterial
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Serologic diagnosis of bacterial
infection Interpreting serologic test data
Acute and convalescence antibody titers
Antibody specificity and cross-reactivity
False-negative and false-positive serologic test results
Value of serologic test:
Population studies
Immune status testing
Congenital infections
Infection after the newborn period
Haemaglutination Inhibition
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Haemaglutination Inhibition
Pengenceran 1/8 1/16 1/32 1/64 1/128 1/256 1/512
serum
fase akut
Konvalesen
= Haemaglutination
= Inhibition of haemaglutination
Haemaglutination Inhibition
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Haemaglutination Inhibition
Pengenceran 1/8 1/16 1/32 1/64 1/128 1/256 1/512
serum
fase akut
Konvalesen
= Haemaglutination
= Inhibition of haemaglutination
Antibody detection methods and
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y
applications
Particle agglutinations assays Direct, natural particle agglutination Indirect, carrier particle agglutination
Precipitations assays Double immunodiffusion Counterimmunoelectrophoresis Flocculation
Complement fixation test Neutralization test
Antistreptolysin O test T. pallidum immobilization
Complement Fixation Test
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Complement Fixation Test
Serum + Test AG & Complement sRBC Hemolysis+(negative)
No HemolysisSerum + Test AG & Complement
sRBC+ (positive)
Neutralization test
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Neutralization test
T. pallidum immobilization, antibody will
immobilize movement
Antistreptolysin O test, antibody will
neutralize streptolysin O, prevent
hemolysis.
Antibody detection methods and
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y
applications Microscope-assisted labeled reagent techniques
Indirect fluorescent antibody test
Fluorescence antibody tests with enhanced sensitivity
Immunoassay with labeled reagents
Enzyme immunoassay
Other immunoassay
Western blotting
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Rapid methods and automation in the
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microbiology laboratory
Microscopic methods for rapid detection Rapid biochemical test performed on
isolated colonies from solid media Rapid enzymatic test using chromogenic
substrate
Reference
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Mahon, C.R., Manuselis Jr, G., 1995.
Diagnostic Microbiology, W.B. Saunders
Company.
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Thank you