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In Germany, biological agents are classified by
Mannitol salt agar - Staphylococcus
MacConkey agar – Enterobacteriaceae
What is Susceptibility testing?
• Susceptibility testing is used to determine which antimicrobials will inhibit the growth of bacteria causing a specific infection
• Results from this test will help a healthcare practitioner determine which drugs are likely to be most effective in treating an infection
What is a Minimum Inhibitory Concentration (MIC) assay
Used to determine the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism
The Gradient diffusion method
Etest gradient diffusion method
• Employs thin plastic test strips that are impregnated on underside with a dried antibiotic concentration gradient and are marked on upper surface with a concentration scale
• MIC is determined by the intersection of the lower part of the ellipse shaped growth inhibition area with test strip • Etest results correlate well with MICs
• however, some systematic biases toward higher or lower MICs
The Modified Kirby Bauer method
Disk diffusion test
Modified Kirby Bauer method is a routine technique to test sensitivity of isolates in clinical labs
• disc diffusion method was originally described in 1966
• well standardised and has been widely evaluated
• Limitations
• does not provide accurate information about the minimum inhibitory concentration (MIC)
• does not provide reliable results with some antibiotic/organism combinations
• e.g. penicillin G in Neisseria meningitidis and Streptococcus pneumoniae
What does MIC stand for?
minimum inhibitory concentration
Immune-based tests
Enzyme-Linked Immuno Spot assay – ELI-Spot
Can use method to look for antibodies, measure immune molecules (e.g. cytokines) and immune cells (e.g. T cells)
ELI-Spot can be used to determine chronic or recent Borrelia burgdorferi infections
• measures reactive T cells to Borrelia Ags – much more sensitive than ELISA
• Other bacterial infections ELI-Spot can be used for include
• Chlamydia pneumonia & Chlamydia trachomatis
M. tuberculosis (IFN-gamma)
Yersinia species
What is Serotyping?
Based on observation that strains of the same species can differ in their antigenic determinants
• Classic tool for identifying bacterial strains (or serotypes)
• Applied to numerous bacteria that express different serotypes
Salmonella
Pseudomonas aeruginosa
E. coli
Shigella
Yersinia
Vibrio cholera
Immune-based techniques
Genotypic methods
What is Multiplex PCR (mPCR)?
In diagnostic laboratories the use of PCR is often limited by cost and availability of adequate sample volume
• To overcome these issues mPCR uses different pairs of primers to simultaneously amplify multiple regions of the nucleic acid of the sample
• significant benefits in cost, time, and exact diagnosis
• Minimises number of separate reactions
• detect several pathogens at the same time in single specimen e.g. sexually transmitted pathogens
• Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus ducrey
• Technique allows diagnosis of several diseases with a single diagnostic test, with sensitivity, specificity, and speed
• indispensable values in diagnostic tests!
• Limitations such as nonspecific products generated through primer-primer interactions may interfere with amplification of targets
• decreasing sensitivity and selectivity of reactions
What is Loop-mediated isothermal amplification (LAMP-PCR)?
Alternative to PCR-based methodologies in clinical labs
• uses 4-6 primers recognizing 6-8 distinct regions of target DNA for a highly specific amplification reaction
• strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification
• significant increase in detection limits, efficiency, selectivity, and specificity
• Used to detect species that cause chorioamnionitis and infections associated with premature labor, Ureaplasma parvum and Ureaplasma urealyticum
What revolutionised molecular diagnostics and gold standard for detection and quantification of bacterial pathogens (also viral pathogens)?
Real-time PCR
… can directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA
Digital PCR – dPCR steps
as ddPCR shows good sensitivity to
low-abundance DNA this method is
as ddPCR shows good sensitivity to low-abundance DNA this method is used to diagnose a variety of bacterial infections that are sometimes hard to identify, due to stage i.e. pathogen concentration very low
• Borrelia burgdorferi, Staphylococcus, Listeria and Mycobacterium tuberculosis • direct count of bacterial number
• improve sensitivity for detection of TB
Fluorescent in situ hybridisation – FISH
• Binding of short fluorescence-labelled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents → fluorescence microscopy
• allows identification of bacterial pathogens at genus or species level
• provides information about spatial resolution, morphology and identification of key pathogens in mixed species sample
• Used for detecting bacteria in biopsies and blood cultures (i.e. sepsis)
• Now automated systems which speeds up process
What are Molecular assays for antimicrobial resistance (AMR) doing?
Molecular AMR diagnostics detect resistance-coding genes or resistance-associated mutations in DNA extracted from purified bacterial isolates or directly from clinical samples
Much faster results than phenotypic methods
Global Antimicrobial Resistance Surveillance System (GLASS)
What are Point of care tests (POCTs) - biosensors?
Need for rapid diagnosis has resulted in the development of novel diagnostic devices based upon the detection and quantification of specific analytes by biorecognition elements
• colorimetric/fluorimetric or electrochemical
• implemented in community, primary care and hospital settings
Different forms:
POCTs – immune (antibody)-based
POCTs – nucleic acid-based
POCTs – protein and glycan-based (Detection of microbial antigens as indicators of infection by specific pathogens, Alternative is the use of host biomarkers to distinguish classes of infecting microbes)
Rapid evaporative ionization mass spectrometry (REIMS)
• Quickly and accurately speciate bacteria based upon their species-specific lipid profile
• Developing customised platform that enables automated, high-throughput REIMS analysis
• minimal user input and operation and is suitable for use in clinical microbiology labs
• The ‘iknife’
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