Diagnostic Microbiology. dr. Agus Eka Darwinata, S.Ked., Ph.D

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1 Diagnostic Microbiology dr. Agus Eka Darwinata, S.Ked., Ph.D

2 symptoms Overview Microbiologic epidemiology Patient s sign and Pathogen identification is essential for effective antimicrobial and supportive theraphy Antimicrobial empirical treatment base on:

3 microorganism Definitive microbiologi diagnostic: Direct microscopic visualization Cultivation and identification Detection of microbial antigens Detection of microbial DNA or RNA Detection of host immune response to

4 Patient history and Physical Examination is the most important

5 Clinical Procedures (medical history and physical examination) Patient Laboratory diagnostic test and precedures All laboratory studies must be directed by the patient s history and physical examination

6 History and examination Serology EXAMPLES OF SPECIMENS Urinary tract infection (UTI) Midstream urine Wound Pus or swab Meningitis CSF & blood Pyrexia of unknown origin (PUO) Blood for culture + serology Pneumonia Sputum, lavage, serology Differential diagnosis Nucleic acid detection method EXAMPLES OF MOLECULAR TECHNIQUES DNA hybridization Nucleic acid amplification testing Polymerase chain reaction (PCR) Ligase chain reaction (LCR) Automated DNA amplification Real time PCR Clinical specimens Microscopy Culture TYPES OF MIA Enrichment Selective Solid Liquid Atmosphere Aerobic CO 2 Anaerobic Microaerophilic Cell culture Virus Chlamydia MICROSCOPY Light Direct (stool parasites) Gram (CSF bacteria) Z N (sputum TB) Giemsa (blood malaria) Immunofluorescence Respiratory syncytial virus (RSV) diagnosis Electron microscopy (EM) Virus detection and identification Zone of inhibition Bacterial growth Susceptibility Identification Typing Public health measures 37ºC Filter paper disc Diagnosis Treatment EXAMPLES OF TYPING METHODS Phage Serology Bacteriocin Endonuclease digestion Multilocus sequence typing Whole genome sequence EXAMPLES OF SEROLOGICAL TECHNIQUES Agglutination Precipitation Complement fixation Virus neutralization Enyzme linked immunosorbent assay (ELISA) Radioimmunoassay (RIA) Immunofluorescence

7 Direct Visualization of Microorganism

8 Direct Visualization of Microorganism pathogenic organisms (excluding viruses) can often be directly visualized by microscopic examination of patient specimens. can provide the first screening step in arriving at a specific identification. The organisms to be examined do not need to be alive or able to multiply. Microscopy yields rapid and inexpensive results, allow the clinician to initiate treatment without waiting for the results of a culture.

9 Gram Stain The most common and useful staining procedure is the Gram stain, which separates bacteria into two classifications according to their cell wall composition. More about Gram stain:

10 Intepretation Shape and Gram ex. Gram-negative rod, Gram-positive coccus

11 organisms/ml. Gram stain applications: The Gram stain is important therapeutically because gram-positive and gram-negative bacteria differ in their susceptibility to various antibiotics, be used to guide initial therapy until the microorganism can be definitively identified. Gram stain limitations: The number of microorganisms required is relatively high. Visualization with the Gram stain requires greater than 10 4

12 Acid-fast stain S t a i n s s u c h a s Z i e h l - Neelsen are used to identify organisms that have waxy material (mycolic acids) in their cell walls. The most clinically important acid-fast bacterium is Mycobacterium tuberculosis, which appears pink, often beaded, and slightly curved Mycobacterium tuberculosis stained with acid-fast stain

13 India ink preparation Useful in detecting Cryptococcus neoformans in CSF One drop of centrifuged CSF is mixed with one drop of India ink on a microscope slide beneath a glass cover slip. Cryptococci are identified by their large, transparent capsules that displace the India ink particles. India ink preparation of Cryptococcus neoformans in cerebrospinal fluid

14 Potassium hydroxide preparation (KOH) Treatment with potassium hydroxide (KOH) dissolves host cells and bacteria, sparing fungi One drop of sputum or skin scraping is treated with 10 percent KOH, and the specimen is examined for fungal forms. Fungi in unstained nasal sinus exudate

15 GROWING BACTERIA IN CULTURE Culturing is routine for most bacterial and fungal infections. Microorganisms isolated in culture are identified using such characteristics as colony size, shape, color, Gram stain, hemolytic reactions on solid media, odor, and metabolic properties. Pure cultures provide samples for antimicrobial susceptibility testing The success of culturing depends on appropriate collection and transport techniques and on selection of media. appropriate culture

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17 MAC. A B A, Lactose-fermenting, gram-negative rods producing pink colonies on MacConkey (MAC). B, Nonlactose-fermenting, gram-negative rods producing colorless colonies on

18 A B A, Lactose-fermenting Escherichia/Citrobacter-like organisms growing on MacConkey (MAC). Notice the dry appearance of the colony and the pink precipitate of bile salts extending beyond the periphery of the colonies. B, Close-up of dry, flat Escherichia/Citrobacter-like lactose fermenters growing on MAC

19 A A, Klebsiella/Enterobacter-like lactose fermenters growing on MacConkey (MAC). Notice the pink, heaped, mucoid appearance. B, Close-up of Klebsiella/Enterobacter-like colonies on MAC. B Notice the mucoid, heaped appearance and the slightly creamcolored center after 48 hours growth.

20 rods. Left, blood agar plate (BAP): small, white colonies are grampositive cocci. Right, BAP: large, gray, mucoid colonies are enteric gram-negative

21 Illustration of form or margin to describe colonial morphology

22 Illustration of elevations to describe colonial morphology

23 Swarming colonies of Proteus spp. The organism was inoculated in the middle of the blood agar plate (arrow)

24 Diphtheroid colonies with rough edges, dry appearance, and umbonate center growing on blood agar plate, buff color.

25 Color In contrast to pigmentation, color is a term used to describe a particular genus in genera Coagulase-negative staphylococci are white Enterococcus spp. may appear gray Certain Micrococcus spp. and Neisseria (nonpathogenic) spp. are yellow or off-white Diphtheroids are buff. Most gram-negative rods are gray on BAP.

26 Example of white colonies of coagulase-negative staphylococci on blood agar plate

27 Example of the yellow colonies characteristic of certain nonpathogenic species of Neisseria organisms on blood agar plate

28 Pigment P. aeruginosa green, sometimes a metallic sheen Serratia marcescens brick-red, especially at room temperature Kluyvera spp. blue Chromobacterium violaceum purple Prevotella melaninogenica brown-black (anaerobic) Pigment production for these organisms is variable.

29 A,Pseudomonas aeruginosa illustrating the metallic sheen and green pigmentation of colonies on blood agar plate (BAP). B, Not all strains of the same organism have the same colonial appearance. This is a mucoid strain of P. aeruginosa on BAP.

30 Brick-red pigment of Serratia marcescens, which is evident on MacConkey (right). This brick-red pigment should not be confused with lactose fermentation. The pigment is slightly visible on chocolate (left). Additional incubation at room temperature enhances the brick-red pigmentation.

31 Brick-red pigment of Serratia marcescens, which is evident on MacConkey (right). This brick-red pigment should not be confused with lactose fermentation. The pigment is slightly visible on chocolate (left). Additional incubation at room temperature enhances the brick-red pigmentation.

32 Odor S. aureus old sock (stocking that has been worn c ously for a few days without washing); this odor is evident when growing on mannitol salt agar P. aeruginosa fruity or grapelike P. mirabilis putrid Haemophilus spp. musty basement, mousy or mouse nest smell Nocardia field spp. freshly plowed

33 Identification of Bacteria

34 Single-enzyme tests

35 Automated system

36 Tests based on the presence of metabolic pathways Rapid manual biochemical system for bacterial identification. Different appearances of the upper and lower pairs of wells indicate the positive or negative ability of a bacterium to utilize each substrate.

37 Serum antigens or antibodies reaction. Latex agglutination test A. Schematic representation of antigens agglutinating latex beads with bound antibody. B. Photograph of agglutination

38 Principle of enzyme-linked immunosorbent assay (ELISA)

39 Nucleic acid amplification for diagnosis PCR: Band patterns appearing in lanes 1-4 are specific for the cytotoxin genes (vvha) of V. vulnificus

40 Applications: Nucleic acid amplification techniques are generally quick, easy, and accurate. A major use of these techniques is for the detection of organisms that cannot be grown in vitro or for which current culture techniques are insensitive. Limitation: the occurrence of false-positives due to cross-contamination with other microorganisms nucleic acid. PCR tests are often costly and require skilled personnel.

41 SUSCEPTIBILITY TESTING After a pathogen is cultured, its sensitivity to specific antibiotics serves as a guide in choosing antimicrobial therapy Disk-diffusion method

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44 Terima Kasih