Chapter 12Chapter 12

Detection and Identification of Microorganisms

ObjectivesObjectives

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 methods that are used to type bacterial strains in epidemiological investigations.

Target Microorganisms for Target Microorganisms for Molecular-Based TestingMolecular-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

Applications of Molecular Based Applications of Molecular Based Testing in Clinical MicrobiologyTesting in Clinical Microbiology

Rapid or high-throughput identification of microorganisms

Detection and analysis of resistance genes

Genotyping Classification Discovery of new microorganisms

Specimen CollectionSpecimen Collection

Preserve viability/nucleic acid integrity of target microorganisms

Avoid contamination Appropriate time and site of collection (blood,

urine, other) Use proper equipment (coagulant, wood, or

plastic swab shafts) Commercial collection kits are available The Clinical and Laboratory Standards Institute

(CLSI) has guidelines for proper specimen handling

Sample PreparationSample Preparation

Consider the specimen type (stool, plasma, CSF)

More rigorous lysis procedures are required to penetrate cell walls

Consider the number of organisms in the sample

Inactivate inhibitors (acidic polysaccharides in sputum or polymerase inhibitors in CSF)

Inactivate RNases

PCR Detection of PCR Detection of Microorganisms: Quality ControlMicroorganisms: Quality Control

PCR and other amplification methods are extremely sensitive and very specific. For accurate test interpretation, use proper controls. Positive control: positive template Negative control: negative template Amplification control: omnipresent

template unrelated to target Reagent blank: no template present

PCR Quality Control: Internal PCR Quality Control: Internal ControlsControls

Homologous extrinsic Controls for

amplification

Heterologous extrinsic Controls for extraction

and amplification

Heterologous intrinsic Human gene control

Target sequence

Quality Control: False PositivesQuality Control: False Positives

Contamination: check reagent blank Dead or dying organisms: retest 3–6

weeks after antimicrobial therapy Detection of less than clinically significant

levels

Quality Control: False PositivesQuality Control: False Positives

Improper collection, specimen handling Extraction/amplification failure: check

internal controls Technical difficulties with chemistry or

instrumentation: check method and calibrations

Antimicrobial AgentsAntimicrobial 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 action3. chemical structure

Sites of Action of Antimicrobial Sites of Action of Antimicrobial AgentsAgents

Mechanisms for Development of Mechanisms for Development of Resistance to Antimicrobial AgentsResistance to Antimicrobial Agents

Enzymatic inactivation of agent Altered target Altered transport of agent in or out Acquisition of genetic factors from other

resistant organisms

Advantages of Molecular Detection of Advantages of Molecular Detection of Resistance to Antimicrobial AgentsResistance to Antimicrobial Agents

Mutated genes are strong evidence of resistance

Rapid detection without culturing Direct comparison of multiple isolates in

epidemiological investigations

Molecular EpidemiologyMolecular Epidemiology

Epidemic: rapidly spreading outbreak of an infectious disease

Pandemic: a disease that sweeps across wide geographical areas

Epidemiology: collection and analysis of environmental, microbiological, and clinical data

Molecular EpidemiologyMolecular Epidemiology

Phenotypic analysis measures biological characteristics of organisms.

Molecular epidemiology is a genotypic analysis targeting genomic or plasmid DNA. Species, strain, or type-specific DNA

sequences are the sources of genotype information.

O = Outbreak strain

1-6 = Isolates

= Changes from

outbreak strain

Pulsed-field Gel Electrophoresis Pulsed-field Gel Electrophoresis (PFGE)(PFGE)

M O 1 2 3 4 5 6

M O 1 2 3 4 5 6

Criteria for PFGE Pattern Criteria for PFGE Pattern Interpretation: Rule of ThreeInterpretation: 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 2–3 Test isolate is closely related to the outbreak strain.

Possibly related 2 4–6 Test isolate is possibly related to the outbreak strain.

Different >3 >6 Test isolate unrelated to the outbreak.

*Compared to the outbreak strain.

Arbitrarily Primed PCR: Random Arbitrarily Primed PCR: Random Amplification of Polymorphic DNA (RAPD)Amplification of Polymorphic DNA (RAPD)

M = Molecular weight markerO = Outbreak strainFour isolates differ from the outbreak strain.

M O

Interspersed Repetitive ElementsInterspersed 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

Other Genotypic Methods Used to Other Genotypic Methods Used to Type OrganismsType Organisms

Plasmid fingerprinting with restriction enzymes

RFLP analysis Amplified Fragment Length Polymorphism

(AFLP) Interspersed repetitive elements Ribotyping spa typing Multilocus sequence typing

Comparison of Molecular Comparison of Molecular Epidemiology MethodsEpidemiology Methods

Method Typingcapacity

Discriminatory

power

Reproducibility Ease ofuse

Ease of interpretation

Plasmid analysis

Good Good Good High Good

PFGE High High High Moderate Goodmoderate

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 Good–high

VirusesViruses

“Classical methods” of detection include antibody detection, antigen detection, or culture.

Molecular methods of detection include target, probe, and signal amplification.

Tests are designed for identification of viruses, determination of viral load (number of viruses per ml of fluid), and genotyping by sequence analysis.

Test Performance Features for Test Performance Features for Viral Load MeasurementViral 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

Viral GenotypingViral Genotyping

Viral genes mutate to overcome antiviral agents.

Gene mutations are detected by sequencing.

Primary resistance mutations affect drug sensitivity but may slow viral growth.

Secondary-resistance mutations compensate for the primary-resistance growth defects.

SummarySummary

Molecular-based methods offer sensitive and direct detection of microorganisms.

Due to high sensitivity and specificity, proper quality control is critical for molecular testing.

Several molecular methods are used to type bacterial strains in epidemiological investigations.

Target, probe, or signal amplification procedures are also used to determine viral load.