Identification of Microorganism
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Transcript of Identification of Microorganism
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Chapter 12
Detection and Identification of Microorganisms
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Objectives
Identify the advantages and disadvantages of
using molecular-based methods as compared to
traditional culture-based methods.
Explain the value of controls, in particular
amplification controls, in ensuring the reliability
of PCR results.
Compare and contrast the molecular methodsthat are used to type bacterial strains in
epidemiological investigations.
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Target Microorganisms for
Molecular-Based Testing
Those that are difficult or time-consuming to
isolate
e.g.,Mycobacteria
Hazardous organisms
e.g.,Histoplasma, Coccidiodes
Those without reliable testing methods
e.g.,HIV, HCV
High-volume tests
e.g.,S. pyogenes, N.gonorrhoeae, C. trachomatis
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Applications of Molecular Based
Testing in Clinical Microbiology
Rapid or high-throughput identification of
microorganisms
Detection and analysis of resistancegenes
Genotyping
Classification Discovery of new microorganisms
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Specimen Collection
Preserve viability/nucleic acid integrity of targetmicroorganisms
Avoid contamination
Appropriate time and site of collection (blood,urine, other)
Use proper equipment (coagulant, wood, orplastic swab shafts)
Commercial collection kits are available The Clinical and Laboratory Standards Institute
(CLSI) has guidelines for proper specimenhandling
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Sample Preparation
Consider the specimen type (stool, plasma,
CSF)
More rigorous lysis procedures are required topenetrate cell walls
Consider the number of organisms in the
sample
Inactivate inhibitors (acidic polysaccharides insputum or polymerase inhibitors in CSF)
Inactivate RNases
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PCR Detection of
Microorganisms: Quality Control
PCR and other amplification methods are
extremely sensitive and very specific. For
accurate test interpretation, use propercontrols.
Positive control: positive template
Negative control: negative templateAmplification control: omnipresent
template unrelated to target
Reagent blank: no template present
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PCR Quality Control: Internal
Controls
Homologous extrinsic
Controls for
amplification
Heterologous
extrinsic
Controls for extraction
and amplification Heterologous intrinsic
Human gene control
Target sequence
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Quality Control: False Positives
Contamination: check reagent blank
Dead or dying organisms: retest 36
weeks after antimicrobial therapy Detection of less than clinically significant
levels
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Quality Control: False Positives
Improper collection, specimen handling
Extraction/amplification failure: check
internal controls Technical difficulties with chemistry or
instrumentation: check method and
calibrations
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Antimicrobial Agents
Inhibit growth (-static); e.g., bacteriostatic,
fungistatic
Kill organisms (-cidal); e.g., bacteriocidal,fungicidal, viricidal
Antimicrobial agents are classified by:
1. static/-cidal2. mode of action
3. chemical structure
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Sites of Action of Antimicrobial
Agents
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Mechanisms for Development of
Resistance to Antimicrobial Agents
Enzymaticinactivation of agent
Altered target
Altered transportof agent in or out
Acquisition of genetic factorsfrom other
resistant organisms
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Advantages of Molecular Detection of
Resistance to Antimicrobial Agents
Mutated genes are strong evidence of
resistance
Rapid detection without culturing Direct comparison of multiple isolates in
epidemiological investigations
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Molecular Epidemiology
Epidemic: rapidly spreading outbreak of
an infectious disease
Pandemic: a disease that sweeps acrosswide geographical areas
Epidemiology: collection and analysis of
environmental, microbiological, andclinical data
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Molecular Epidemiology
Phenotypic analysis measures biological
characteristics of organisms.
Molecular epidemiology is a genotypicanalysis targeting genomic or plasmid
DNA.
Species, strain, or type-specific DNAsequences are the sources of genotype
information.
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O = Outbreak strain
1-6 = Isolates
= Changes fromoutbreak strain
Pulsed-field Gel Electrophoresis
(PFGE)
M O 1 2 3 4 5 6
M O 1 2 3 4 5 6
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Criteria for PFGE Pattern
Interpretation: Rule of Three
Category Genetic
differences*
Fragment
differences*
Epidemiological
interpretation
Indistinguishable 0 0 Test isolate is the same
strain as the outbreak
strain.Closely related 1 23 Test isolate is closely
related to the outbreak
strain.
Possibly related 2 46 Test isolate is possibly
related to the outbreakstrain.
Different >3 >6 Test isolate unrelated
to the outbreak.
*Compared to the outbreak strain.
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Arbitrarily Primed PCR: Random
Amplification of Polymorphic DNA (RAPD)
M = Molecular weight marker
O = Outbreak strain
Four isolates differ from the outbreak strain.
M O
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Interspersed Repetitive Elements
GCC G/T GATGNCG G/A CG C/T NNNNN G/A CG C/T CTTATC C/A GGCCTAC
.GTGAATCCCCAGGAGCTTACATAAGTAAGTGACTGGGGTGAGCG.REP sequence inverted repeat
ERIC sequence inverted repeat
PCR amplification priming outward from repetitive elements
generates strain-specific products.
Is the unknown (U) strain A or B?
Isolate A
Isolate B
M A B M A B U
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Other Genotypic Methods Used to
Type Organisms
Plasmid fingerprinting with restrictionenzymes
RFLP analysis
Amplified Fragment Length Polymorphism(AFLP)
Interspersed repetitive elements
Ribotyping spatyping
Multilocus sequence typing
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Comparison of Molecular
Epidemiology Methods
Method Typing
capacity
Discriminatory
power
Reproducibility Ease of
use
Ease of
interpretation
Plasmid
analysis
Good Good Good High Good
PFGE High High High Moderate Good
moderate
Genomic
RFLP
High Good Good High Moderate
poor
Ribotyping High High High Good High
PCR-RFLP Good Moderate Good High High
RAPD High High Poor High Good
high
AFLP High High Good Moderate High
Repetitive
elements
Good Good High High High
Sequencing High High High Moderate Goodhigh
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Viruses
Classical methods of detection include
antibody detection, antigen detection, orculture.
Molecular methods of detection includetarget, probe, and signal amplification.
Tests are designed for identification of
viruses, determination of viral load(number of viruses per ml of fluid), andgenotyping by sequence analysis.
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Test Performance Features for
Viral Load Measurement
Characteristic Description
Sensitivity Lowest level detected at least 95% of the time
Accuracy Ability to determine true value
Precision Reproducibility of independently determined test
results
Specificity Negative samples are always negative and positive
results are true positives
Linearity A serial dilution of standard curve closely
approximates a straight line
Flexibility Accuracy of measurement of virus regardless of
sequence variations
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Viral Genotyping
Viral genes mutate to overcome antiviralagents.
Gene mutations are detected bysequencing.
Primary resistance mutationsaffect drugsensitivity but may slow viral growth.
Secondary-resistance mutationscompensate for the primary-resistancegrowth defects.
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Summary
Molecular-based methods offer sensitive anddirect detection of microorganisms.
Due to high sensitivity and specificity, properquality control is critical for molecular testing.
Several molecular methods are used to typebacterial strains in epidemiological
investigations. Target, probe, or signal amplification
procedures are also used to determine viralload.