Laboratory Diagnosis of Bacterial Infection

Osman Sianipar Department of Clinical Pathology Gadjah Mada University, Yogyakarta

Sept 28, 2006

Lab diagnosis of bacterial infection

Clinical Pathology

Clinical Chemistry Hematology Serology Microbiology

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Lab diagnosis of bacterial infection

Lab Diagnosis

Culture and Identification Antigen detection Antibody detection

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Lab diagnosis of bacterial infection

Introduction

Microbiologist have relied on culture isolation of microorganism to establish the etiology of bacterial infection. 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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Lab diagnosis of bacterial infection

Introduction

Some limitations to classic approach of culture isolation 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 care decision on the basis of culture results. Some effort have been done in order to solve these limitations.

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Culture and Identification

Source of specimen Transport media Culture technique:

Automatic/manual Media Colony morphology Staining, microscopic examination Biochemistry Serologic technique

Identification:

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Antigen detection

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Lab diagnosis of bacterial infection

Antigen detection methods

Particle agglutination: agglutination: Particle Latex Latex agglutination agglutination Staphylococcal Staphylococcal co-agglutination co-agglutination Liposome-mediated Liposome-mediated agglutination agglutination Precipitin Precipitin tests: tests: Tube Tube and and agar agar precipitin precipitin Counter-immuno-electrophoresis Counter-immuno-electrophoresis Microscope-assisted Microscope-assisted labeled-antibody labeled-antibody staining: staining: Fluorescence-labeled Fluorescence-labeled antibody antibody Enzyme-labeled Enzyme-labeled antibody antibody

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Antigen detection methods

Solid-phase Solid-phase immunoassay immunoassay with with labeled labeled

reagent: reagent: Enzyme Enzyme immunoassay immunoassay Fluorescence Fluorescence immunoassay immunoassay Radioimmunoassay Radioimmunoassay

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Particle agglutination

Y Y +
Patient serum (antigen)

Y Y
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Agglutination

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Antibody structure

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Particle agglutination
Antibody (IgG (IgG or or IgM; IgM; polyclonal polyclonal or or monoclonal) monoclonal) Antibody Reaction Reaction depends depends on: on: Particle Particle size size Avidity Avidity of of antibody antibody Antibody Antibody type type (polyclonal/monoclonal) (polyclonal/monoclonal) pH pH and and ionic ionic strength strength of of the the test test specimen specimen Reaction Reaction temperature temperature Antigen Antigen concentration concentration

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Antibody production

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Particle agglutination
Particle Particle can can be: be: Latex Latex Formalin-killed Formalin-killed S. S. aureus aureus (protein (protein A) A) Liposome 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)

AG well

AB well

Antigen Precipitin band Antibody Agar

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Counterimmunoelctrophoresis
(Precipitin tests)
Agar coated Glass slide AG well AB well

Cathode (-)

AG migration

AB migration

Anode (+)

Buffer chamber
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Fluorescence-labeled antibody

F F

Direct
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Indirect
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Fluorescence-labeled antibody

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Enzyme-labeled antibody

E E

Direct
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Indirect
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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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EIA, Inhibition

Conc

OD

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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

Van der Velden. Leukemia 2003 (www)

Antibody detection

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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

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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

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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

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Antibody detection methods and applications

Particle agglutinations assays

Direct, natural particle agglutination Indirect, carrier particle agglutination Double immunodiffusion Counterimmunoelectrophoresis Flocculation

Precipitations assays

Complement fixation test Neutralization test

Antistreptolysin O test T. pallidum immobilization

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Complement Fixation Test

Serum +

Test AG & Complement + sRBC

Hemolysis (negative)

Serum + Test AG & Complement + sRBC

No Hemolysis (positive)

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Neutralization test

T. pallidum immobilization, antibody will immobilize movement Antistreptolysin O test, antibody will neutralize streptolysin O, prevent hemolysis.

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Antibody detection methods and applications

Microscope-assisted labeled reagent techniques

Indirect fluorescent antibody test Fluorescence antibody tests with enhanced sensitivity Enzyme immunoassay Other immunoassay

Immunoassay with labeled reagents

Western blotting

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Rapid methods and automation in the microbiology laboratory

Microscopic methods for rapid detection Rapid biochemical test performed on isolated colonies from solid media Rapid enzymatic test using chromogenic substrate

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Reference

Mahon, C.R., Manuselis Jr, G., 1995. Diagnostic Microbiology, W.B. Saunders Company.

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Thank you

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