BASIC DIAGNOSIS.ppt

Baedah Madjid DEPART. OF MICROBIOLOGY, MEDICAL

FACULTY, HASANUDIN UNIVERSITY2007

The student must:The student must:

• know what methods are used to determine the know what methods are used to determine the

microbial cause of the diseasemicrobial cause of the disease

• understand how these methods are combines with understand how these methods are combines with

clinical examination and other investigations in clinical examination and other investigations in

clinical practice.clinical practice.

• recognize the limit of laboratory-base evidence in recognize the limit of laboratory-base evidence in

making a specific microbiological diagnosis.making a specific microbiological diagnosis.

DEFENITIVE MICROBIOLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

RATIONAL THERAPY

CONTROL

DEFENITIVEDIAGNOSIS OF INFECTIOUS DISEASES

CLINICAL FEATURES RADIOGRAPH

LABORATORY DIAGNOSIS

PATHOLOGY MICROBIOLOGY CLINICAL CHEMISTRY

ACCURATE MICROBIOLOGY DIAGNOSIS

SPECIMENS

METHODS

INSTRUMENTS/REAGENTS

HUMAN RESOURCES

METHODS :METHODS : → high sensitivity & high → high sensitivity & high specificityspecificity- High sensitivity & High specificity :High sensitivity & High specificity :

- - High sensitivity & Low specificity:High sensitivity & Low specificity:

all infected individuals detected, but no false-all infected individuals detected, but no false-positive responsespositive responses

all infected individuals detected, but many false-all infected individuals detected, but many false-positives response.positives response.

- - Low sensitivity & High specificity:Low sensitivity & High specificity:not all infected individuals detected (some false-not all infected individuals detected (some false-negatives) , but no false-positivesnegatives) , but no false-positives

- - Low sensitivity & Low specificity:Low sensitivity & Low specificity:not all infected individuals are detected (some false-not all infected individuals are detected (some false-negatives) and some who response are falsely positive.negatives) and some who response are falsely positive.

INSTRUMENTS:INSTRUMENTS: valid valid

Depend on:

REAGENTS:REAGENTS: fresh made fresh made

- Expert in his job- Expert in his job - High dedicated- High dedicated

HUMAN RESOURCESHUMAN RESOURCES

SPECIEMENS:SPECIEMENS: appropriate appropriateSputum, faces, blood, etcSputum, faces, blood, etc

Specimen should appropriatelySpecimen should appropriately- CollectedCollected- StoredStored- TransportedTransported- ProcessedProcessed

Specimen amount:Specimen amount: sufficient for the test sufficient for the test

Collecting MethodCollecting Method: Sterile: Sterile

Time of specimen collection:Time of specimen collection: punctual punctual

- Before antimicrobial therapy - Before antimicrobial therapy - Stage of the course of the diseaseStage of the course of the disease- Any time, or early morning, or 24 hours Any time, or early morning, or 24 hours

Location:Location: the site of suspected infection the site of suspected infection

Specimen container:Specimen container: sterile & screw cap sterile & screw cap

Time:Time:

●●Specimen should be processed not more Specimen should be processed not more than 8 hr.than 8 hr.● ● Specimen (in transport medium) Specimen (in transport medium) should be processed in 24 hr.should be processed in 24 hr.

Temperature :emperature : depend on bacteria depend on bacteria suspected: 4suspected: 4ooC, or in room temperatureC, or in room temperature

Specimen should be transported to Specimen should be transported to Laboratory as soon after collection, in:Laboratory as soon after collection, in:

- Transport medium- Transport medium- Room temperature or ice- Room temperature or ice

SPECIMEN

BACTERIA MACROMOLECULE

Bottle/med.transport

DirectPreparation

Isolation & IdentificationMicroscopy

Ag AbDNA OR

RNA

SPECIMEN

DIRECT SMEAR

WET MOUNT STAINING

FLUORESCENCE ACID FASTG R A M

Light microscopy:1. Wet mount: - morphology - motility 2. Preparation a. Gram stain: morphology, Gram positive or negative b. Acid fast stain: morphology, Acid fast bacilli c. Fluorescence: Increasing sensitivity of Acid fast

preparation.

SPECIMEN

ENRICHMENT MEDIUM

BOTTLE OR TRANSPORT MEDIUM

SELECTIVE/DIFFERENTIAL MEDIUM

PURE CULTURE

Identification GRAM BIOCHEM TESTS DNA Tes RAG

1. Transport medium = carrier medium2. Basic medium 3. Enriched medium4. Enrichment medium5. Selective medium6. Differential medium7. Special medium8. Anaerobic medium

1. Colony appearance 2. Gram: Morphology & Gram characteristic3. Motality : solid medium or semi-solid4. Biochemical Reaction5. Macromolecule identification : - Ag microbial detection - Ab detection - DNA/RNA detection

Isolated colony → colony appearance

1. Form and margin

2. Sizes

3. Elevation

4. Clarity

5. Color : pigment, chemical reaction

6. Hemolysis : α, ß, & γ

Colony’s color: Red Colorless

Colony’s color : pigment

Non-hemolysisß - Hemolysis

1. Charbohydrate Metabolism

- Test for sugar fermentation, VP-MR Test

2. Protein Metabolism

- Indol test

3. Lipid Metabolism ( rare)

5. Single enzyme test

Catalase Test, coagulase test, oxidase test, urease test

glucose lactose

Triple Sugar Iron Agar

Citrat Medium

DNAse Medium

Invitro Ag-Ab reacton:1. Agglutination Test2. Precipitation & flocculation Test3. Complement fixation Test4. Immuno-fluorescent5. Radio-immuno assay (RIA)6. Enzyme linked immunosoebent assay

(ELISA)7. Blotting immuno assay

1. Direct agglutination :

- Widal

- Gol. Darah

2. Particle agglutination

a. Latex Agglutination Test

b. Co-agglutination Test

c. Hemagglutination (TPHA)

A. Flocculation Test

- VDRL

- RPR

B. Precipitation Test

1. Immune-double diffusion : Ag orAb.

detection

2. Radial immunodiffusion: qualitative &

quantitative.

1. Solid phase ELISA

a. Direct

b. Indirect

Qualitative & quantitative

2. Membrane bound SPIA

Qualitative

1. Gen-Probe (Hybridization)

- Less sensitive

2. Polymerase chain reaction (PCR)

- High sensitivity

● RFLP (Retriction Fragment Length

Polymorphism): polymorphism

● DNA Sequencing

A.Diffusion Test:

● Disc Diffusion test

B. Dilution test:

● Tube Dilution test

● Microbroth Panel Dilution Test

C. Combine : E. Susceptibility Test

Disc Diffusion test:

- R or S“E. Test susceptibility”

MBC & MIC

• MBC: Minimal Bactericidal Concentration

• MIC: Minimal Inhibitory Concentration

Microbroth Panel Test

MBC & MIC