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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