Laboratory Diagnosis of Bacterial Infection

Laboratory Diagnosis of  Bacterial Infection Osman Sianipar  Department of Clinical Pathology Gadj Gadjah ah Mada Mada Univ Univer ersi sity ty,, Yogy Yogyak akar arta ta

Clinical Pathology 

Clinical Chemistry

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Hematology

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Serology

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Microbiology

Clinical Pathology 

Clinical Chemistry

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Hematology

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Serology

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Microbiology

Lab Diagnosis

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Culture and Identification

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 Antigen detection

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 Antibody detection detection

Introduction 

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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.

Introduction 

Some limitations to classic approach of culture isolation and identification:     

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 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.

Culture and Identification   

Source of specimen Transport media Culture technique:  

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 Automatic/manual Media

Identification:    

Colony morphology Staining, microscopic examination Biochemistry Serologic technique

 Antigen  Antigen detection detection

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

 Antigen detection methods  

Solid-phase Solid-phase immunoassay immunoassay with with labeled labeled reagent: reagent:  Enzyme immunoassay immunoassay  Enzyme Fluorescence Fluorescence immunoassay immunoassay   Radioimmunoassay Radioimmunoassay  

Particle agglutination

Y      Y

Y

      Y

Y      Y

+

Y

      Y

Patient serum (antigen)

Agglutination

 Antibody structure

Particle agglutination  Antibody (IgG (IgG or or IgM; IgM; polyclonal polyclonal or or monoclonal) monoclonal)  Antibody Reaction depends depends on: on:  Reaction   

Particle size size Particle  Avidity  Avidity of of antibody antibody   

 Antibody type type (polyclonal/monoclonal) (polyclonal/monoclonal)  Antibody pH and and ionic ionic strength strength of of the the test test specimen specimen  pH   

Reaction temperature temperature Reaction  Antigen concentration concentration  Antigen   

 Antibody production

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

Latex agglutination

Staphylococcal Coagglutination

Liposome-mediated aglutination

 Tube and agar precipitin (Precipitin tests)

AG well

AB well

Antigen Precipitin band  Antibody

Agar 

Counterimmunoelctrophoresis (Precipitin tests) Agar coated  Glass slide AG well

Cathode (-)

AG migration

AB well

AB migration

Buffer chamber 

Anode (+)

Fluorescence-labeled antibody

F F

Direct

Indirect

Fluorescence-labeled antibody

Enzyme-labeled antibody

E E

Direct

Indirect

Enzyme-labeled antibody 

Not require a fluorescent microscope

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Improve sensitivity

Solid-phase immunoassay with labeled reagent 

More amenable to automation

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Includes: 

Enzyme immunoassay

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Fluorescence immunoassay

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Radioimmunoassay

EIA double AB

Conc

OD

EIA, indirect double AB

Conc

OD

EIA, competitive

Conc

OD

EIA, Inhibition

Conc

OD

Membrane-bound solid-phase EIA 

Hybridization technique

PCR 

PCR 

 Antibody detection

Serologic diagnosis of bacterial infection 

Interpreting serologic test data 

 Acute and convalescence antibody titers

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 Antibody specificity and cross-reactivity

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False-negative and false-positive serologic test results

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Value of serologic test: 

Population studies

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Immune status testing

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Congenital infections

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Infection after the newborn period

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

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 applications 

Particle agglutinations assays  

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Precipitations assays   

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

Complement fixation test Neutralization test  Antistreptolysin O test  T. pallidum immobilization 

Complement Fixation Test

Serum +

Test AG & Complement + sRBC

Hemolysis (negative)

Serum + Test AG & Complement + sRBC

 No Hemolysis (positive)

Neutralization test 

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T. pallidum immobilization, antibody will immobilize movement

 Antistreptolysin O test, antibody will neutralize streptolysin O, prevent hemolysis.

 Antibody detection methods and applications 

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Microscope-assisted labeled reagent techniques 

Indirect fluorescent antibody test

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Fluorescence antibody tests with enhanced sensitivity

Immunoassay with labeled reagents 

Enzyme immunoassay

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Other immunoassay

Western blotting

Rapid methods and automation in the microbiology laboratory   

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

Reference 

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