PERANAN MIKROBIOLOGI DALAM

DIAGNOSIS PENYAKIT INFEKSI

dr. Agus Eka Darwinata, Ph.D.

 CLINICAL MICROBIOLOGY
• Clinical microbiology is the discipline of
detection, characterization, and quantification
of microbes from patients in order to enable
diagnosis, management and treatment of
infectious diseases.
What is Clinical Microbiology Laboratory?

Laboratory that provide service:

• Analyzing specimens collected from sick patients.
• Gathering data that are enable the correct diagnosis to
be made for suspected infectious patients.
• Help in guiding the selection of the right antimicrobial
therapy for infectious patients.
• Recognize the emergence of resistance to antimicrobial
agents
• Help in managing infectious diseases outbreaks by
identifying pathogen.
Clinical Microbiology Laboratory Test

Direct Indirect

Detection of Detection
microorganism, antibodies
the structural (serum)
component,
or their product
Microbes (bacterial) Identification
 Microbial Identification
1. Microscopy
• Gram staining
• Ziehl-Neelsen staining (Acid Fast Bacilli)
• KOH (Fungi)
2. Culture
3. Biochemical tests (Species identification)
• Manual
• Semiautomatic
• Automatic
4. Antimicrobial Susceptibility Tests (AST)
5. Molecular tests (for non cultivable and fastidious
bacteria, viruses)
6. Serology (antigen-antibody detection)
Bacterial Smear Preparation
Microscopy: Gram staining
Microscopy: Gram staining result
Microscopy: Ziehl-Neelsen (ZN) staining for AFB
Microscopy: Ziehl Neelsen (ZN) staining for AFB
Microbiological/ Bacterial Culture
• Growing of microorganisms on culture medium
• Aerobic vs. anaerobic
• Usually 35+2°C
• Solid, semisolid, and liquid media
• Solid (agar plate):
– Common: Blood agar
– Selective and differential: MacConkey agar (gram
negative bacilli)
– Enriched: for fastidious bacteria (Thayer Martin (GO);
BCYE agar)
Bacterial Culture: streak technique
Bacterial Culture: Type of bacterial colony
 Bacterial Culture: type of hemolysis
Observed on blood agar
plate.
• Beta (clear zone):
Streptococcus pyogenes
• Alpha (greenish zone):
Streptococcus
pneumoniae, S. mitis, S.
mutans
• Gamma: no hemolysis
 Bacterial Culture: specific
characteristic of bacteria
• Swarming phenomenon:
Proteus sp.

• Pyocyanin pigment:
(green): Pseudomonas
sp.
 Bacterial Culture:
Gram negative colonies on MC agar
 Biochemical Tests
For bacterial identification (species)
• Manual
• Semiautomated
• Automated
 Biochemical Tests: Manual

Time consuming (1 day), full skilled lab analyst,

wrong bacterial ID >
Biochemical Tests: Semiautomated

Read and
match with
database
(computer,
special
software)

Shorter time, < wrong ID than manual

 Biochemical Tests: Automated

Shorter time (6-8h), << wrong ID

than manual, semiautomatic
Vitek 2 Compact
Biochemical Tests: Automated (Vitek 2)
Biochemical Tests:
Automatic (2)

MALDI-TOF
• Mass spectrometry
• Matrix assisted
• Short time (10 min)
• Sensitive and specific
(ID)
• Expensive
 Antimicrobial Susceptibility Tests (AST)

• To predict the outcome of treatment with the

antimicrobial agents tested
• To provide information to the clinician to
guide in selecting appropriate antimicrobial
therapy for a particular clinical problem
 Routine AST Methods
• Broth microdilution
• Automated-instrument method – Vitek,
Phoenix, Microscan, Sensititre..
• Antimicrobial gradient (Etest, MIC Evaluator)
• Disk diffusion method (Kirby-Bauer)
Disk diffusion remains the more accessible and
economic method
 AST METHODS: DISK DIFFUSION
KIRBY - BAUER

• One of the most established and best proven of all susceptibility tests
• Continues to be updated and refined through frequent CLSI publications
• Low cost
• Inherent flexibility in drug selection
• Ability to respond quickly to changes in interpretive breakpoints or when
new agents are available
• No established interpretive criteria for some bacteria
• MIC tests are recommended for some drug/bacterial sp. combination
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AST METHOD: GRADIENT DIFFUSION (E-TEST)

• Commercial methods- follow manufacturer’s

directions
• Same testing procedure as the disk method
• Simple and flexible, may be used to test for fastidious
and anaerobic bacteria
• MICs generally agree well with MICs by standard
broth or agar dilution methods
• More expensive than the disk method

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 AST METHODS: AUTOMATED

• Results are generated in a shorter period (3.5 – 16

hours)
• Computer software used to interpret AST results
• Includes “expert system" for analyzing test results for
atypical patterns and unusual resistance phenotypes
• Lessened ability to detect some types of antimicrobial
resistance
• Cost is higher
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Viral Identification (Detection)
 Viral Infections
Diagnosis (Detection)
 Molecular Microbiology

• Development of new genetically engineered

vaccines, biotechnology, antimicrobial
development, etc.
• Have a direct influence on the clinical practice
of Medical Microbiology
 Application of Molecular Microbiology
• Classification of organism (genotyping).
• Identification and confirmation of isolate obtained from
culture.
• Early detection of pathogens in clinical specimen.
• Rapid detection of antibiotic resistance.
• Detection of mutations.
• Differentiation of toxigenic from non-toxigenic strains.
• For fastidious bacteria or for unculturable microbes (to
culture, grow slowly or present in extremely small
numbers in clinical specimen).
Genotyping (Phylogenetic Tree)
16S rRNA gene identity using BLAST
Carbapenem Resistance Genes Detection

• lane 1, control blaKPC gene;

• lane 2, control blaOXA-
48 gene;
• lane 3, control blaVIM gene;
• lane 4, control blaNDM-
1 gene;
• lane 5, control blaIMP gene;
• lane 6, control blaOXA-
23 gene.
• M, Molecular mass markers
(200 - 1500 bp DNA ladder).
Mutation Detection