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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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
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