Transcript Molecular Diagnostics 6

Molecular Diagnostics
Detection and Identification of
Microorganisms
Chapter 12
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Molecular Diagnostics
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Applications of Molecular Based
Testing in Clinical Microbiology
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Rapid or high-throughput identification of
microorganisms
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Those that are difficult or time-consuming to isolate
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Hazardous organisms
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e.g., Histoplasma, Coccidiodes
Those without reliable testing methods
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e.g., Mycobacteria
e.g., HIV, HCV
High-volume tests
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e.g., S. pyogenes, N. gonorrhoeae, C. trachomatis
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Molecular Diagnostics
Applications of Molecular Based
Testing in Clinical Microbiology
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Detection and analysis of resistance genes
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Genotyping
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mecA  oxacillin resistance in Staphylococcus aureus
vanA, vanB, and vanC  vancomycin resistance in
Enterococcus
katG and inhA  isoniazid resistance in M. tuberculosis
Mycobacterium, HCV, and HIV
Reclassification of microorganisms for
epidemiological purposes, and to predict therapeutic
efficacy
Discovery of new microorganisms
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Molecular Diagnostics
Specimen Collection
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Preserve viability/nucleic acid integrity of target
microorganisms
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Avoid contamination that could yield false-positive results
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Due to the sensitivity of molecular testing
Appropriate time and site of collection (blood, urine, other)
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Viability is not much critical for molecular testing
DNA and especially RNA can be damaged in lysed or nonviable cells
Obtimize the likely presence of the infectious agent
E.g., Salmonella typhi is initially present in peripheral blood but not in
urine or stool until at least 2 weeks after infection
Use proper equipment (coagulant, wood, or plastic swab
shafts)
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E.g., Plastics are less adherent to the microorganisms and will not
interfere with PCR reagents as do emanations from wooden shafted
swabs
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Molecular Diagnostics
Sample Preparation
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Depending on the microorganism more rigorous
lysis procedures may be required
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Mycobacteria and fungi have thick cell walls that are more
difficult to lyse than other bacteria and parasites.
Gram-positive bacteria cell wall is thicker than gramnegative bacteria
Mycoplasma, lacks a cell wall, thus avoid spontaneous
lysis of the cells and loss of nucleic acids
The concentration of organisms within the clinical
sample must be considered.
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Centrifuge to concentrate the fluid and the organisms
within the fluid
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Molecular Diagnostics
Sample Preparation
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Inhibitors of enzymes used in molecular analysis
may be present in clinical specimens
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Acidic polysaccharides in sputum or polymerase inhibitors
in CSF
if RNA is to be analyzed
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inactivation or removal of RNases in the sample and in all
reagents and materials that come into contact with the
sample
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Molecular Diagnostics
Quiz
In order to increase the stringency of a PCR
reaction we need to,
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a)
b)
c)
d)
Decrease the annealing temperature and increase
the annealing time
Increase the annealing temperature and increase
the annealing time
Decrease the annealing temperature and decrease
the annealing time
Increase the annealing temperature and decrease
the annealing time
Molecular Diagnostics
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PCR Detection of
Microorganisms: Quality Control
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PCR and other amplification methods are
extremely sensitive and very specific. For
accurate test interpretation, use proper
controls.
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Positive control: positive template
Negative template control: negative template
Amplification control: omnipresent template
unrelated to target
Reagent blank/contamination control: no
template present
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Molecular Diagnostics
PCR Quality Control: Internal
Controls
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Homologous extrinsic
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wild-type–derived control with a nontarget-derived
sequence insert
Added to every sample after nucleic acid extraction and
before amplification
Amplification occurs using the same primers as for the
target
Good for ensuring that amplification occurs in the sample
does not control for target nucleic acid degradation during
Target sequence
extraction.
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Molecular Diagnostics
PCR Quality Control: Internal
Target sequence
Controls
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Heterologous extrinsic
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Nontarget-derived controls
Added to every sample before nucleic acid
extraction
Will ensure that extraction and amplification
procedures are acceptable
A second set of primers must also be added to the
reaction for this control to be amplified.
The procedure must be optimized such that the
amplification of the control does not interfere with
the amplification of the target.
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Molecular Diagnostics
PCR Quality Control: Internal
Controls
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Heterologous intrinsic
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Eukaryotic genes.
ensures that human nucleic acid is
present in the sample in addition to
controlling for extraction and
amplification
Requires two amplification reactions for
the sample, or the amplification
procedure be multiplexed
Target sequence
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Molecular Diagnostics
Quality Control: False Positives
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Contamination: check reagent blank
Dead or dying organisms: retest 3–6 weeks after
antimicrobial therapy
Detection of less than clinically significant levels
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Molecular Diagnostics
Quality Control: False negative
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Improper collection, specimen handling
Extraction/amplification failure: check internal
controls
Technical difficulties with chemistry or
instrumentation: check method and calibrations
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Molecular Diagnostics
Selection of Sequence Targets
for Detection of Microorganisms
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Molecular Diagnostics
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Molecular Diagnostics
Mechanisms for Development of
Resistance to Antimicrobial Agents
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Enzymatic inactivation of agent
Altered target
Altered transport of agent in or out
Acquisition of genetic factors from other resistant
organisms
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Molecular Diagnostics
Advantages of Molecular Detection
of Resistance to Antimicrobial Agents
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Mutated genes are strong evidence of resistance
Rapid detection without culturing
Direct comparison of multiple isolates in
epidemiological investigations
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Molecular Diagnostics
Molecular Epidemiology
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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
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Molecular Diagnostics
Molecular Epidemiology
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Phenotypic analysis measures biological
characteristics of organisms.
Molecular epidemiology is a genotypic analysis
targeting genomic or plasmid DNA.
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Species, strain, or type-specific DNA sequences are
the sources of genotype information.
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Molecular Diagnostics
Pulsed-field Gel Electrophoresis
(PFGE)
Organisms with large genomes
or multiple chromosomes
DNA is digested with
infrequently cutting restriction
enzymes
Large fragments (hundreds of
thousands of base pairs) are
resolved by PFGE
Patterns of organisms will differ
depending on the chromosomal
DNA sequence of the organisms
O = Outbreak strain
1-6 = Isolates
= Changes from outbreak strain
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Molecular Diagnostics
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
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.
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Molecular Diagnostics
Arbitrarily Primed PCR: Random
Amplification of Polymorphic DNA (RAPD)
MO
M = Molecular weight marker
O = Outbreak strain
Four isolates differ from the outbreak strain.
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Molecular Diagnostics
Interspersed Repetitive Elements
Enterobacterial repetitive intergenic consensus
Repetitive extragenic palindromic
PCR amplification
priming outward from
repetitive elements
generates strain-specific
products.
Is the unknown (U) strain
A or B?
Molecular Diagnostics
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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
Good–high
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Molecular Diagnostics
Viruses
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“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.
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Molecular Diagnostics
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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Molecular Diagnostics
Viral Genotyping
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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.