PERANAN MIKROBIOLOGI DALAM DIAGNOSIS PENYAKIT INFEKSI. dr. Agus Eka Darwinata, Ph.D.

Transcription

1 PERANAN MIKROBIOLOGI DALAM DIAGNOSIS PENYAKIT INFEKSI dr. Agus Eka Darwinata, Ph.D.

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4 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.

5 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.

6 Clinical Microbiology Laboratory Test Direct Indirect Detection of microorganism, the structural component, or their product Detection antibodies (serum)

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8 Microbes (bacterial) Identification

9 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)

10 Bacterial Smear Preparation

11 Microscopy: Gram staining

12 Microscopy: Gram staining result

13 Microscopy: Ziehl-Neelsen (ZN) staining for AFB

14 Microscopy: Ziehl Neelsen (ZN) staining for AFB

15 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)

16 Bacterial Culture: streak technique

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18 Bacterial Culture: Type of bacterial colony

19 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

20 Bacterial Culture: specific characteristic of bacteria Swarming phenomenon: Proteus sp. Pyocyanin pigment: (green): Pseudomonas sp.

21 Bacterial Culture: Gram negative colonies on MC agar

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23 Biochemical Tests For bacterial identification (species) Manual Semiautomated Automated

24 Biochemical Tests: Manual Time consuming (1 day), full skilled lab analyst, wrong bacterial ID >

25 Biochemical Tests: Semiautomated Read and match with database (computer, special software) Shorter time, < wrong ID than manual

26 Biochemical Tests: Automated Vitek 2 Compact Shorter time (6-8h), << wrong ID than manual, semiautomatic

27 Biochemical Tests: Automated (Vitek 2)

28 Biochemical Tests: Automatic (2) MALDI-TOF Mass spectrometry Matrix assisted Short time (10 min) Sensitive and specific (ID) Expensive

29 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

30 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

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32 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 32

33 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 33

34 AST METHODS: AUTOMATED Results are generated in a shorter period ( 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 34

35 Viral Identification (Detection)

36 Viral Infections Diagnosis (Detection)

37 Molecular Microbiology Development of new genetically engineered vaccines, biotechnology, antimicrobial development, etc. Have a direct influence on the clinical practice of Medical Microbiology

38 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).

39 Genotyping (Phylogenetic Tree)

40 16S rrna gene identity using BLAST

41 Carbapenem Resistance Genes Detection lane 1, control bla KPC gene; lane 2, control bla OXA- 48 gene; lane 3, control bla VIM gene; lane 4, control bla NDM- 1 gene; lane 5, control bla IMP gene; lane 6, control bla OXA- 23 gene. M, Molecular mass markers ( bp DNA ladder).

42 Mutation Detection

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