basic diagnosis.ppt
TRANSCRIPT
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Baedah Madjid DEPART. OF MICROBIOLOGY, MEDICAL
FACULTY, HASANUDIN UNIVERSITY2007
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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.
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DEFENITIVE MICROBIOLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES
RATIONAL THERAPY
CONTROL
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DEFENITIVEDIAGNOSIS OF INFECTIOUS DISEASES
CLINICAL FEATURES RADIOGRAPH
LABORATORY DIAGNOSIS
PATHOLOGY MICROBIOLOGY CLINICAL CHEMISTRY
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ACCURATE MICROBIOLOGY DIAGNOSIS
SPECIMENS
METHODS
INSTRUMENTS/REAGENTS
HUMAN RESOURCES
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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.
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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
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Specimen should appropriatelySpecimen should appropriately- CollectedCollected- StoredStored- TransportedTransported- ProcessedProcessed
Specimen amount:Specimen amount: sufficient for the test sufficient for the test
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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
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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
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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
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SPECIMEN
BACTERIA MACROMOLECULE
Bottle/med.transport
DirectPreparation
Isolation & IdentificationMicroscopy
Ag AbDNA OR
RNA
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SPECIMEN
DIRECT SMEAR
WET MOUNT STAINING
FLUORESCENCE ACID FASTG R A M
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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.
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SPECIMEN
ENRICHMENT MEDIUM
BOTTLE OR TRANSPORT MEDIUM
SELECTIVE/DIFFERENTIAL MEDIUM
PURE CULTURE
Identification GRAM BIOCHEM TESTS DNA Tes RAG
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1. Transport medium = carrier medium2. Basic medium 3. Enriched medium4. Enrichment medium5. Selective medium6. Differential medium7. Special medium8. Anaerobic medium
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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
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Isolated colony → colony appearance
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1. Form and margin
2. Sizes
3. Elevation
4. Clarity
5. Color : pigment, chemical reaction
6. Hemolysis : α, ß, & γ
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Colony’s color: Red Colorless
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Colony’s color : pigment
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Non-hemolysisß - Hemolysis
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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
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glucose lactose
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Triple Sugar Iron Agar
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Citrat Medium
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DNAse Medium
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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
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1. Direct agglutination :
- Widal
- Gol. Darah
2. Particle agglutination
a. Latex Agglutination Test
b. Co-agglutination Test
c. Hemagglutination (TPHA)
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A. Flocculation Test
- VDRL
- RPR
B. Precipitation Test
1. Immune-double diffusion : Ag orAb.
detection
2. Radial immunodiffusion: qualitative &
quantitative.
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1. Solid phase ELISA
a. Direct
b. Indirect
Qualitative & quantitative
2. Membrane bound SPIA
Qualitative
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1. Gen-Probe (Hybridization)
- Less sensitive
2. Polymerase chain reaction (PCR)
- High sensitivity
● RFLP (Retriction Fragment Length
Polymorphism): polymorphism
● DNA Sequencing
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A.Diffusion Test:
● Disc Diffusion test
B. Dilution test:
● Tube Dilution test
● Microbroth Panel Dilution Test
C. Combine : E. Susceptibility Test
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Disc Diffusion test:
- R or S“E. Test susceptibility”
MBC & MIC
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• MBC: Minimal Bactericidal Concentration
• MIC: Minimal Inhibitory Concentration
Microbroth Panel Test
MBC & MIC