12. Mikro klinik

8/8/2019 12. Mikro klinik

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Chapter 12

Diagnosing InfectionsTiana Milanda


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Diagnosis of Microbial Infection

Patient Clinicaldiagnosis

HaematologyBiochemistry

Non-microbiologicalinvestigations

Radiology

Sample Take the correct specimen

Take the specimen correctly

Label & package thespecimen up correctly

Appropriate transport &storage of specimen


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SpecimenCollection

The success of identification andtreatment dependson how specimensare collected,handled, and stored

General asepticprocedures must beused


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Diagnosis of Microbial Infection

2. Culture

on plates or in broth

identification by biochemical orserological tests on pure growth

from single colony

1. Microscopy

Decolorise CounterstainStain

unstained or stained with e.g.Gram stain

5. Sensitivities

3. Imunologicalmethod

4. Genotypicmethod

by disc diffusionmethods,

breakpoints or

MICs


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1. MicroscopyUnstained preparations

Wet prep Dark-ground illumination

for syphilis


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1. MicroscopyStained preparations

Gram-stain Acid-fast stain

Ziehl-Neelsen


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1. MicroscopyStained preparations

Special structures Fluorescence

Direct, e.g. auramine Immunofluorescence


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2. Culture of Bacteria

Solid media Agar plates

For Identification For Enumeration

Slopes For safe long-term

culture, e.g. Lowenstein-Jensen media for TB Liquid media (broth)

For enrichment or

maximum sensitivity


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Advantages of Solid Media

isolation of singleclonal colonies get bacterium in

pure culture

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2

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http://www.aims.org.au/nmlsw/nmlsw_jpg/index.php?directory=.&currentPic=6


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Advantages of Solid Media identify by colonial

morphology


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Advantages of Solid Media

quantification by colony-forming units :

Pengenceran

Pengenceran

Hitung koloni

menggunakan alatcoulter counter darimasing-masing cawanpetri, kalikan denganfaktor pengenceran,lalu rata-ratakan. Hasilyang baik 30-300koloni per cawan petri

Contoh :30.10 1 X 35.10 2 x 40.10 3

3=bakteri/ml

1 ml

1 ml 1 ml 1 ml

1 ml 1 ml

Sampel padat

Sampel kental

9 mlmediumcair

kocok

20 ml medium padat


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2. Culture : Physiological/BiochemicalCharacteristics

Diagnostic tests for determining the presenceof specific enzymes and assessing nutritionaland metabolic activities

Examples Fermentation of sugars Capacity to metabolize complex polymers Production of gas Presence of enzymes


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Identification of Bacteria


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3. Immunological Methods

Characteristics of antibodies can reveal thehistory of a patients contact withmicroorganisms or other antigens

Serology : the branch of immunology thattraditionally deals with in vitro diagnostictesting of the serum

Serological testing Serotyping


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General Features of Immune Testing

Strategies Agglutination Precipitation Immunodiffusion Complement fixation Fluorescent antibody tests Immunoassay tests

Specificity and sensitivity


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Agglutination and PrecipitationReactions

Agglutination antigens are whole cells suchas red blood cells or bacteria with

determinant groups on the surface Precipitation the antigen is a soluble

molecule


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Agglutination Testing Antibodies cross-link the antigens to form visible

clumps Performed routinely to determine ABO and Rh blood

types

Widal test : tube agglutination test for diagnosingsalmonelloses

Rapid plasma regain (RPR) test : tests for antibodies tosyphilis

Weil-Felix reaction : diagnoses ricketsial infections Latex agglutination tests : tiny latex beads withantigens affixed


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Visualizing Antigen-AntibodyInteractions


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Precipitation Tests

The soluble antigen is precipitated by anantibody

Reaction is observable as a cloudy or opaquezone at the point of contact


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Visualizing Antigen-AntibodyInteractions


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Immunodifussion

Double diffusion method Immunoelectrophoresis


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The Western Blot for DetectingProteins

Involves electrophoretic separation of proteinsfollowed by an immunoassay to detect thoseproteins

Test material is electrophoresed in a gel toseparate out particular bands Gel transferred to a special blotter that binds the

reactants in place Blot developed by incubating it with a solution of

antigen or antibody labeled with radioactive,fluorescent, or luminescent labels


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Complement Fixation

Lysin or cytolysin : an antibody that requirescomplement to complete the lysis of itsantigenic target cell


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Flurorescent Antibodies andImmunofluorescence Testing

Direct testing : an unknown test specimen orantigen is fixed to a slide and exposed to afluorescent antibody solution of knowncomposition

Indirect testing : the fluorescent antibodiesare antibodies made to react with the Fc

region of another antibody


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Immunoassays

Extremely sensitive methods that permit rapidand accurate measurement of trace antigen orantibody

Radioactive isotope labels Enzyme labels Sensitive electronic sensors


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Radioimmunoassay (RIA)

Antibodies or antigens labeled with a radioactiveisotope used to pinpoint minute amounts of acorresponding antigen or antibody

Compare the amount of radioactivity present in asample before and after incubation with aknown, labeled antigen or antibody

Large amounts of a bound radioactive componentindicate that the unknown test substance was notpresent


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Radioimmunoassay (RIA)


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Enzyme-Linked Immunosorbent Assay(ELISA)

Enzyme-antibody complex that can be used asa color tracer for antigen-antibody reactions

Indirect Direct


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4.Genotypic Methods

Primary advantage over phenotypic methods:actually culturing the microorganisms is notalways necessary

Also are increasingly automated with resultsobtained very quickly


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4. Genotypic Methods DNA Analysis Using Genetic Probes

Hybridization- can identify a bacterial species byanalyzing segments of its DNA

Small fragments of single-stranded DNA or RNA called

probes Known to be complementary to the specificsequences of DNA from a particular microbe Unknown test DNA from cells is bound to blotter paper Add probes to blotter

Observe for signs that the probes have become fixed to thetest DNA

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labeled probe

genomic DNA G A T C A G T A G

C T A G T C A T C


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Nucleic Acid Sequencing and rRNAAnalysis

Comparison of the sequence of nitrogen basesin rRNA (16s rRNA and 18s rRNA)

Effective for differentiating general groupdifferences

Can be fine-tuned to identify at the specieslevel


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Prokaryote vs eukaryote ribosomes


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Advancements to sequencing

Fluorescent tagging sequence data Computer read & analyzed


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Polymerase Chain Reaction

Rapid identification of pathogens Developed for a wide variety of bacteria,

viruses, protozoa, and fungi


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The PCR procedureTargetsequence

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Genomic DNA

Cycle 1yields

2molecules

Cycle 2

yields4

molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

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3

3

5

Primers

Newnucleo-tides

3

APPLICATION With PCR, any specific segment the targetsequence within a DNA sample can be copied many times(amplified) completely in vitro .

TECHNIQUE The starting materials for PCR are double-stranded DNA containing the target nucleotide sequence to becopied, a heat-resistant DNA polymerase, all four nucleotides,

and two short, single-stranded DNA molecules that serve asprimers. One primer is complementary to one strand at one endof the target sequence; the second is complementary to theother strand at the other end of the sequence.

RESULTS During each PCR cycle, the target DNAsequence is doubled. By the end of the third cycle, one-fourthof the molecules correspond exactly to the target sequence,with both strands of the correct length (see white boxesabove). After 20 or so cycles, the target sequence moleculesoutnumber all others by a billionfold or more.

Denaturation:Heat brieflyto separateDNA strands

1

Annealing:Cool to allowprimers tohydrogen-bond.

2

Extension:DNA polymeraseadds nucleotidesto the 3 end ofeach primer

3


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Sensitivities test

Antimicrobial sensitivity MIC and MBC


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www.themegallery.com


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MIC and MBC

Minimum Inhibitory Concentration

MIC -Lowest Concentration of antimicrobic which will inhibitin vitro growth of microorganism.

Minimum Bacteriocidal ConcentrationMBC -Lowest Concentration of antimicrobic which will kill amicroorganism in vitro.


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h l h d


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Broth Dilution Method

Macro dilution Method- Using test tube- Media : 1-5 mL/tube

Micro dilution Method- Using 96-wells microtiter plates- Media : 0.1 0.2 mL/well

12. Mikro klinik

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