Identification of Microorganism

8/12/2019 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.

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Identification of Microorganism