Chapter 12

Detection and Identification of Microorganisms

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

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

Applications of Molecular Based Testing in Clinical Microbiology

Rapid or high-throughput identification of microorganisms Detection and analysis of resistance genes Genotyping Classification Discovery of new microorganisms

Specimen 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 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 Microorganisms: 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 Controls

Homologous extrinsic
Controls for amplification
Target sequence

Heterologous extrinsic
Controls for extraction and amplification

Heterologous intrinsic
Human gene control

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

Quality Control: False Positives

Improper collection, specimen handling Extraction/amplification failure: check internal controls Technical difficulties with chemistry or instrumentation: check method and calibrations

Antimicrobial Agents
Inhibit growth (-static); e.g., bacteriostatic, fungistatic Kill organisms (-cidal); e.g., bacteriocidal, fungicidal, viricidal Antimicrobial agents are classified by: 1. static/-cidal 2. mode of action 3. chemical structure

Sites of Action of Antimicrobial Agents

Mechanisms for Development of Resistance 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 Resistance to Antimicrobial Agents

Mutated genes are strong evidence of resistance Rapid detection without culturing Direct comparison of multiple isolates in epidemiological investigations

Molecular 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 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.

Pulsed-field Gel Electrophoresis (PFGE)

M O 1 2 3 4 5 6

O = Outbreak strain 1-6 = Isolates

M O 1 2 3 4 5 6

= Changes from outbreak strain

Criteria for PFGE Pattern Interpretation: Rule of Three

Category
Indistinguishable

Genetic differences*
0

Fragment differences*
0

Epidemiological interpretation
Test isolate is the same strain as the outbreak strain.

Closely related

23

Test isolate is closely related to the outbreak strain.

Test isolate is possibly related to the outbreak strain. Test isolate unrelated to the outbreak.

Possibly related

46

Different

>3

>6

*Compared to the outbreak strain.

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

MO

M = Molecular weight marker O = Outbreak strain Four isolates differ from the outbreak strain.

Interspersed Repetitive Elements

REP sequence inverted repeat
.GTGAATCCCCAGGAGCTTACATAAGTAAGTGACTGGGGTGAGCG.

ERIC sequence inverted repeat PCR amplification priming outward from repetitive elements generates strain-specific products.
Isolate A

GCC G/T GATGNCG G/A CG C/T NNNNN G/A CG C/T CTTATC C/A GGCCTAC

Isolate B M A B M A B U

Is the unknown (U) strain A or B?

Other Genotypic Methods Used to Type 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 Epidemiology Methods

Method Plasmid analysis PFGE Genomic RFLP Ribotyping PCR-RFLP RAPD AFLP Repetitive elements Sequencing Typing capacity Good High High High Good High High Good High Discriminatory power Good High Good High Moderate High High Good High Reproducibility Good High Good High Good Poor Good High High Ease of use High Moderate High Good High High Moderate High Moderate Ease of interpretation Good Good moderate Moderate poor High High Goodhigh High High Goodhigh

Viruses
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 Viral Load Measurement

Characteristic Sensitivity Accuracy Precision Specificity Linearity Flexibility Description Lowest level detected at least 95% of the time Ability to determine true value Reproducibility of independently determined test results Negative samples are always negative and positive results are true positives A serial dilution of standard curve closely approximates a straight line Accuracy of measurement of virus regardless of sequence variations

Viral 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.

Summary
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.