molecular diagnostics 1 detection and identification of microorganisms chapter 12

Molecular DiagnosticsMolecular Diagnostics

11

Detection and Identification of Microorganisms

Chapter 12

Molecular DiagnosticsMolecular Diagnostics22

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


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


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


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


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



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


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


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



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.


Target sequence



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


Target sequence


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


Quality Control: False negative

Improper collection, specimen handling Extraction/amplification failure: check internal

controls Technical difficulties with chemistry or

instrumentation: check method and calibrations


Selection of Sequence Targetsfor Detection of Microorganisms


Molecular DiagnosticsMolecular Diagnostics1515 Molecular DiagnosticsMolecular Diagnostics1515


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)

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


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.


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

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

M O


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


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


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 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 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.

molecular diagnostics 1 detection and identification of microorganisms chapter 12

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