Clinical Microbiology Flashcards
ILOs
Understand classical approaches employed in the clinical microbiology laboratory
Be aware of current advancements in approaches to clinical microbiology
Leading types of HCAIs
- UTIs - 34%
- Surgical site infections - 22%
Why study cause of infection?
Prognosis
Treatment
Isolation
Care
How is the cause of an infection determined? Patient Specimin
-
Rapid tests and immunoassays
- Bacteria and fungi: biochemical identification
- Bacteria, fungi, protozoa and viruses: ELISA, flow cytometry, complement fixation
2. Microscopy
- Light: bacteria, fungi, protozoa
- Electron: viruses
3. Culture - Bacteria and fungi: purify and amplify
- Viruses: cytopathology
4. Biochemical Tests
- Bacteria and fungi: identification and sensitivity
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Molecular Testing
- Bacteria, fungi, protozoa, viruses: nucleic acid amplification, sequencing, fingerprinting
How is the cause of an infection determined? 1. Historical workflow
Historical workflow:
- stain-based methodologies for classification of microscopic morphology to support early diagnostic and therapeutic decisions
- microbial culture for propagation of the offending organism on agar or in liquid medium
- biochemical or antigenic techniques for the subsequent metabolic and phenotypic analysis of the microorganism, ultimately leading to microbe identification
- antimicrobial susceptibility testing to confirm therapeutic choices or tailor therapy
How is the cause of an infection determined? 2. Stains for microscopy - Gram Stain
The Gram stain
– Classic stain for differentiating between Gram –ve and Gram +ve bacteria
How is the cause of an infection determined? Stains for microscopy - Acid Fast Stain
• Acid fast stain for tuberculosis
– Also known as the Ziehl- Neelson stain
• Advantages – Specific
– No need for culture
– Performed directly on sputum
How is the cause of an infection determined? Stains for microscopy - PAS (periodic acid-Schiff)
• PAS (periodic acid-Schiff)
– Stains for glycoproteins, often used for fungi
• Disadvantages
– High background
How is the cause of an infection determined? Microbial culture
Selective media
– E.g. Mannitol salt agar, used for the isolation of Staphylococci.
Differential media
– MacConkey agar, recovery of Enterobacteriaceae.
How is the cause of an infection determined? Biochemical techniques - G+
How is the cause of an infection determined? Biochemical Techniques - G-
How is the cause of an infection determined? Biochemical techniques: API strips
• Typically carried out using API strips (Analytical profile index)
– 20 biochemical tests simultaneously
How is the cause of an infection determined? Agglutination assays
- Doesn’t require culture
- Used frequently for detection of viral infections
- Conversely lack of agglutination in some assays is the measure of infection
- visualise as a change in turbidity of solution
How is the cause of an infection determined?
ELISA – Enzyme Linked ImmunoSorbent Assay
Direct and indirect ELISA
Capture ELISA
Direct ELISA
Indirect ELISA
Capture (sandwich) ELISA
Determining antibiotic resistance/susceptibility
Etest strips. Antibiotic present at a gradient of concentrations
- enables quantitative analysis of plates
Modern Clinical microbiology
Modern Clinical microbiology
- Molecular methods (Nucleic acid based)
– NAATs
– NGS
- Mass spectrometric methods
– MALDI TOF
– ESI
Modern Clinical microbiology - Molecular Methods
(Nucleic acid based)
Single and multiplex PCR
– Often no need for culture
– Doesn’t detect live or dead
– Cheap!
– Sensitive
– No gold standard for comparison
– Prone to error in set up
Next gen sequencing
Next generation sequencing of whole bacterial genomes
– Culture dependent currently – Species identification
– Wealth of information
– Becoming cheaper
Next gen seq – Metagenomics and community profiling
– Culture independent
– Total DNA isolated from a sample for metagenomics
– Certain regions such as 16s r RNA sequenced in community profiling
– ‘culturomics’ borne out of limitations
Mass Spec
• Mass spectrometric methods
– MALDI TOF
– ESI
– Cheaper than sequencing
– Reliant on databases of known patterns
