BASIC PRINCIPLES OF

DIAGNOSTIC OF INFECTIOUS
DISEASES
 LEARNING OBJECTIVE
The student should:
 know what methods are used to determine
the microbial cause of the disease
 understand how these methods are combines
with clinical examination and other investi-
gations in clinical practice.
 recognise the limit of laboratory-base eviden-
ce in making a specific microbiological
diagnosis.
 RATIONAL THERAPY

DEFENITIVE MICROBIOLOGICAL
DIAGNOSIS OF INFECTIOUS DISEASES

CONTROL
CLINICAL FEATURES RADIOGRAPH

DEFENITIVE
DIAGNOSIS OF INFECTIOUS DISEASES

LABORATORY DIAGNOSIS

CLINICAL
PATHOLOGY MICROBIOLOGY
CHEMISTRY
 SPECIMENS METHODS

ACCURATE
MICROBIOLOGY DIAGNOSIS

INSTRUMENTS HUMAN
/REAGENTS RESOURCES
 METHODS : → high sensitivity & high
specificity

- High sensitivity & High specificity : all

infected individuals detected, but no false-
positive responses
- High sensitivity & Low specificity: all
infected individuals detected, but many
false-positives response.
- Low sensitivity & High specificity: not all
infected individuals detected (some false-
negatives) , but no false-positives
 - Low sensitivity & Low specificity: not all infected
individuals are detected (some false-negatives)
and some who response are falsely positive.

 INSTRUMENTS: valid

 REAGENTS: fresh made

 HUMAN RESOURCES
- Expert in his job
- High dedicated

 SPECIMENS: Appropriate
Sputum, faces, blood, etc
 APPROPRIATE SPECIMEN
Specimen should appropriately
- collected
- stored
- transported
- processed
Specimen amount: sufficient for the test
 PROPERLY COLLECTED
SPECIMEN
 Sterile method
 Time of specimen collection: punctual
- antimicrobial therapy
- stage of the course of the disease
- any time, or early morning, or 24 hours
 Location: the site of suspected infection
 SPECIMEN STORING
 Specimen container: sterile & screw cap
 Specimen should be processed not more than
8 hr.
 Specimen (in transport medium) should be
processed in 24 hr.
 Temperature : depend on bacteria suspected:
4oC, or in room temperature..
 If necessarily use transport medium
 SPECIMEN TRANSPORT

Specimen should be transported to

Laboratory as soon after collection, in
- Transport medium
- Room temperature or ice
BACTERIOLOGIC IDENTIFICATION
SPECIMEN

BACTERIA MACROMOLECULE

Direct Bottle/med.
Preparation Ag Ab DNA OR
transport RNA

Microscopy Isolation & Identification

A. DIRECT EXAMINATION
SPECIMEN

DIRECT SMEAR

STAINING
WET MOUNT

GRAM ACID FAST FLUORESCENCE

 DIRECT EXAMINATION
Light microscopy:
• 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.
Gram Stain
Acid Fast Stain
Fluorescence Stain
 ISOLATION OF BACTERIA
SPECIMEN

BOTTLE OR TRANSPORT MEDIUM

ENRICHMENT MEDIUM

SELECTIVE/DIFFERENTIAL MEDIUM

PURE CULTURE

Identification GRAM BIOCHEM TESTS AG DNA Tes R

 Media
1. Transport medium = carrier medium
2. Basic medium
3. Enriched medium
4. Enrichment medium
5. Selective medium
6. Differential medium
7. Special medium
8. Anaerobic medium
Inoculation on solid medium
 B. Identification
1. Colony appearance
2. Gram: Morphology & Gram characteristic
3. Motality : solid medium or semi-solid
4. Biochemical Reaction
5. Macromolecule identification :
- Ag microbial detection
- Ab detection
- DNA/RNA detection
Isolated colony → colony
appearance
Colony appearance

1. Form anf margin

2. Sizes
3. Elevation
4. Clarity
5. Color : pigment, chemical reaction
6. Hemolysis : α, ß, & γ
Colony appearance
Colony’s color
Red Colorless
Colony’s color : pigment
ß - Hemolysis & non-hemolysis
Pure Culture
Biochemical reaction

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
Sugar fermentation tests
glucose lactose
Triple Sugar Iron Agar:
Sugars fermentation & protein
metabolism
Citrate as an energy sources
DNAse test
Ag or Ab detection

Invitro Ag-Ab reacton:

1. Agglutination Test
2. Precipitation & flocculation Test
3. Complement fixation Test
4. Immuno-fluorescent
5. Radio-immuno assay (RIA)
6. Enzyme linked immunosoebent assay (ELISA)
7. Blotting immuno assay
Agglutination Test

1. Direct agglutination :
- Widal
- Gol. Darah
2. Particle agglutination
a. Latex Agglutination Test
b. Co-agglutination Test
c. Hemagglutination (TPHA)
Precipitation and flocculation test
A. Flocculation Test
- VDRL
- RPR
B. Precipitation Test
1. Immune-double diffusion : Ag orAb.
detection
2. Radial immunodiffusion: qualitative &
quantitative.
ELISA
(Enzym-linked immunosorbent Assay)
1. Solid phase ELISA
a. Direct
b. Indirect
Qualitative & quantitative
2. Membrane bound SPIA
Qualitative
DNA or RNA detection

1. Gen-Probe (Hybridization)
- Less sensitive

2. Polymerase chain reaction (PCR)

- High sensitivity