PERANAN MIKROBIOLOGI DALAM DIAGNOSIS PENYAKIT INFEKSI

dr. Agus Eka Darwinata, Ph.D.

CLINICAL MICROBIOLOGY

• Clinical microbiology is the discipline ofdetection, characterization, and quantificationof microbes from patients in order to enablediagnosis, management and treatment ofinfectious 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 microorganism, the structural component,

or their product

Detection antibodies

(serum)

Microbes (bacterial) Identification

Microbial Identification1. Microscopy

• Gram staining• Ziehl-Neelsen staining (Acid Fast Bacilli)• KOH (Fungi)

2. Culture3. 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 hemolysisObserved 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 TestsFor bacterial identification (species)

• Manual• Semiautomated• Automated

Biochemical Tests: Manual

Time consuming (1 day), full skilled lab analyst, wrong bacterial ID >

Biochemical Tests: Semiautomated

Shorter time, < wrong ID than manual

Read and match with database

(computer, special

software)

Biochemical Tests: Automated

Vitek 2 CompactShorter time (6-8h), << wrong ID

than manual, semiautomatic

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

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• 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

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 – 16hours)

• 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