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Molecular DiagnosticsMolecular Diagnostics
11
Detection and Identification of Microorganisms
Chapter 12
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Applications of Molecular Based Testing in Clinical Microbiology
Rapid or high-throughput identification of microorganisms 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
Detection and analysis of resistance genes mecA oxacillin resistance in Staphylococcus aureus vanA, vanB, and vanC vancomycin resistance in
Enterococcus katG and inhA isoniazid resistance in M. tuberculosis
Genotyping Mycobacterium, HCV, and HIV
Reclassification of microorganisms for epidemiological purposes, and to predict therapeutic efficacy
Discovery of new microorganisms
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Specimen Collection Preserve viability/nucleic acid integrity of target
microorganisms Viability is not much critical for molecular testing DNA and especially RNA can be damaged in lysed or nonviable cells
Avoid contamination that could yield false-positive results Due to the sensitivity of molecular testing
Appropriate time and site of collection (blood, urine, other) 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) 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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Sample Preparation
Depending on the microorganism more rigorous lysis procedures may be required 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 gram-
negative 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. Centrifuge to concentrate the fluid and the organisms
within the fluid
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Sample Preparation
Inhibitors of enzymes used in molecular analysis may be present in clinical specimens Acidic polysaccharides in sputum or polymerase inhibitors
in CSF if RNA is to be analyzed
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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Quiz
In order to increase the stringency of a PCR reaction we need to,
a) Decrease the annealing temperature and increase the annealing time
b) Increase the annealing temperature and increase the annealing time
c) Decrease the annealing temperature and decrease the annealing time
d) Increase the annealing temperature and decrease the annealing time
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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 proper controls. 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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PCR Quality Control: Internal Controls
Homologous extrinsic 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
extraction. Target sequence
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PCR Quality Control: Internal Controls
Heterologous extrinsic 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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Target sequence
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PCR Quality Control: Internal Controls
Heterologous intrinsic 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
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Target sequence
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Quality 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
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Quality Control: False negative
Improper collection, specimen handling Extraction/amplification failure: check internal
controls Technical difficulties with chemistry or
instrumentation: check method and calibrations
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Selection of Sequence Targetsfor Detection of Microorganisms
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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
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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 across wide geographical areas
Epidemiology: collection and analysis of environmental, microbiological, and clinical data
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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.
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Pulsed-field Gel Electrophoresis (PFGE)
Organisms with large genomes or multiple chromosomes
Organisms with large genomes or multiple chromosomes
DNA is digested with infrequently cutting restriction
enzymes
DNA is digested with infrequently cutting restriction
enzymes
Large fragments (hundreds of thousands of base pairs) are
resolved by PFGE
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
Patterns of organisms will differ depending on the chromosomal DNA sequence of the organisms
O = Outbreak strain1-6 = Isolates = Changes from outbreak strain
O = Outbreak strain1-6 = Isolates = Changes from outbreak strain
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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 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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Arbitrarily Primed PCR: Random Amplification of Polymorphic DNA (RAPD)
M = Molecular weight markerO = Outbreak strainFour isolates differ from the outbreak strain.
M O
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Interspersed Repetitive Elements
PCR amplification priming outward from repetitive elements generates strain-specific products.
Is the unknown (U) strain A or B?
Repetitive extragenic palindromicRepetitive extragenic palindromic
Enterobacterial repetitive intergenic consensusEnterobacterial repetitive intergenic consensus
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Comparison of Molecular Epidemiology 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
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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.
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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 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.