Diagnostic Flashcards
What are diagnostics tests
-laboratory tests
-imagining tests
-endoscopy
+history and clinical exam
What is an aseptic technique
➢ any method used to sterilise and maintain the sterility of an object or location, such as an operating theatre or laboratory, though it may also refer to wound-care to prevent infection
➢ “aseptic” refers to a procedure that is performed under sterile conditions, to avoid contamination
during sampling
Principles for sample collection
- Specimens must be obtained aseptically from a site that is representative of the disease process
- A sufficient quantity of material should be collected
- Specimens must be collected prior to antibiotics to maximise pathogen recovery;
- If cultures are not immediately initiated after collection,
specimens should be refrigerated
Transport and storage of samples
Transport: stop bugs from dying
– get the sample to the lab fast!
– use “transport medium” ex. Amies, charcoal, Cary-Blair
• Storage: only do so if necessary
– 4oC better than anything cooler or
warmer
– long-term storage at -80oC or below
direct microscopic observation advantages
Cost effective and rapid
• Provides immediate information on:
- presence or absence of bacteria, fungi;
- number of organisms
• Allows presumptive identification of bacteria:
- Morphologic characteristics
- Gram-staining properties of microorganisms
• Provides information on the host cellular response (e.g. purulent
inflammatory response is suggestive of bacterial infection)
Candida albicans yeasts an
Staphylococcus shape
Staphyle=grape like
Streptococcus shape
Streptos=twisted
Specific staining methods for fungal identification
-KOH 10% with blue Parker ink
(examination of plucked hair)
• KOH acts as a clearing agent
• When Parker ink is added, the
fungal spores take up the ink and
appear blue-violet
Microscopic examination viruses
-fairly low sensitivity
-not very specific
-expensive equipment
Différent methods to identify an unknown pathogen in clinical specimens
-culture based methods
-non-culture based methods
What is culture based microbial detection for bacteria
Bacterial culture aims to achieve:
1. Isolation of the organism in pure culture
→inoculating the specimen onto appropriate artificial culture
media, followed by
2. Identification of the isolate by:
➢ Microscopic examination
➢ Biochemical reactions
➢ State-of-the art techniques (e.g., MALDI-TOF)
3. Antimicrobial Susceptibility Testing (AST)
After growing the organism in pure culture.
Isolation bacteria
• Most pathogenic bacteria grow on agar
plates.
• Most common contaminants also grow on
agar plates.
• Specific enrichment and selection protocols
can be used to “hunt” for particular
pathogens.
• Different incubation conditions, e.g. aerobic
and anaerobic
Enrichment media
general purpose media supplemented by
blood or other special nutrients to
encourage the growth of fastidious
organisms
Differential media
distinguish between different groups of bacteria on the basis of their biological characteristics
Selective media
favour the growth of
particular microorganisms and inhibit the
growth of others.
Blood agar plates and use
Identification of bacteria
-some bacteria secrete enzymes/toxins that lyse the erythrocytes in the agar, producing zones of haemolysis
Steps in gram staining
Fixation-> Crystal Violet-> Iodine Treatment-> Decolorisation-> Counter stain with Safranin
Oxidase test
The OXIDASE test detects the presence of cytochrome C oxidase. This
enzyme reduces tetramethyl-p-phenylene-diamine (TMPD), a redox dye, to yield
a purple product.
Oxidase +ve
= dark
blue/purple
colour
Oxidase -ve
colourless
Use of biochemical tests to determine the presence or absence of specific
metabolic enzymes in an isolate: by performing a series of such tests,
characteristic patterns of reactivity emerge, providing an identity for the
isolate,
CATALASE TEST
Detects presence of Catalase Enzyme
• If bacteria have the catalase enzyme,
external hydrogen peroxide is
broken down to oxygen and water.
H2O2 -> H2O + O2
• Oxygen is then seen as “bubbles”
Phenotypic traits used for identification:
Oxygen requirements: aerobic/anaerobic growth
• Culture characteristics:
Colonial morphology (size, pigmentation, haemolysis)
• Staining properties:
– Gram stain
– Acid fast (Ziehl Neelsen stain)
– Others, e.g. Polychrome methylene blue (demonstrates
capsule presence) for Anthrax (Bacillus anthracis)
• Microscopic morphology:
Shape, size, cellular arrangement, presence of spores
• Biochemical reactions (or enzyme activities) e.g. Catalase,
Oxidase, Urease
• MALDI-TOF (mass spectrometry)
AST
Antimicrobial susceptibility testing
Microbiology laboratory susceptibility
testing to various antimicrobial agents
• Many bacteria can have unpredictable
susceptibilities to antimicrobial agents
(Gram-negative bacteria)
• Antimicrobial susceptibility of isolates can
be measured “in vitro” to help guide the
selection of the most appropriate
antimicrobial agent
Qualitative methods:
➢ Disc Diffusion:
- A growth medium (Mueller-Hinton
Agar, MHA) is seeded throughout the
plate with the isolate of interest
- Discs impregnated with
antimicrobial agents are dispensed
- After an overnight incubation, the
bacterial growth around each disc is
observed
AST interpretation
SUSCEPTIBLE – the microorganism is inhibited by a concentration of antimicrobial agent that can be attained in blood with the normally recommended dose and implies that an
infection caused by this microorganism may be appropriately treated with the antimicrobial agent.
• RESISTANT - the microorganism is resistant to concentrations of the antimicrobial agent that can be attained with normal doses and implies that an infection caused by this microorganism could not be successfully treated with this antimicrobial agent.
• INTERMEDIATE - a buffer zone to avoid misinterpretation;
treatment is possible if the infection is in body ites where the
antimicrobial is concentrated.
Culture based viruses
• Viruses are obligate intracellular
organisms (grow inside living cells)
– Tissue cell cultures
– Embryonated eggs
– Experimental animals
• Need to keep virus alive in transport
• Specialist lab to grow cells
• Need to inhibit bacterial growth
• Not all viruses grow in cell culture
• Takes a long time, labour intensive
Isolation and characterisation of viruses
Some viruses clump red blood cells, stopping them from settling at the bottom of a well
Culture based microbial detection- FUNGI
Clinical specimens for fungal culture:
• Plucked hairs from the lesions
• Toothbrush
• Skin scrapings
• Exudates
• Biopsies and tissues (histopathology)
Why and when can you use UV fluorescence
Ultraviolet (UV) fluorescence
• Some fungi produce metabolites that
fluorescence of a vivid apple-green
colour when examined under UV light
How can you use antigen-antobody reactions
Diagnostic tests that use:
➢ A KNOWN ANTIGEN (viral/bacterial) to detect specific antibodies in animal serum (serology)
e.g. Enzyme-Linked Immunosorbent Assays (ELISAs)
➢ KNOWN ANTIBODIES (an immunoglobulin -
Ig), to detect bacterial/viral antigens in clinical
samples
e.g. ImmunoFluorescent Antigen (IFA) tests
Serological testing antigen-antibody reactions
Serological tests detect the presence of antibodies (i.e. of an immune
response or Ab titer) in a patient’s serum
➢ Antibodies are specifically produced against certain antigens, making them
useful to diagnose specific bacterial/viral infections
➢ Detecting the host immune response to an infection can indicate:
➢ Ongoing infection (seroconversion) and/or
➢ Past exposure to the pathogen
➢ Maternally derived antibodies
➢ Vaccination
Non-culture based microbial detection-
Detection of nucleic acids (PCR)
Polymerase Chain Reaction (PCR)
revolutionised Science and Diagnostics
• Advantages:
– Very sensitive
– Detects microorganisms that are non viable,
uncultivable or slow growing
– Faster results compared to culture methods
•Limitations: failing to differentiate organisms liv
- Susceptibility of PCR reaction to inhibitors,
Direct detection PCR
Increasingly used, especially for detection
of uncultivable/slow growing organisms
e.g.
• Mycoplasmas
• Mycobacteria
• Spirochetes
• Viruses
• Rapid detection and confirmation of
MRSA/MRSP
Rapid Molecular
Single-step, cartridge-based molecular test devices target
pathogens implicated in clinical syndromes, e.g.:
➢ Respiratory infections
➢ Neurological infections
➢ Gastroenteritis
WHAT IS POINT OF CARE TESTING
Point-of-Care Testing (POCT) is clinical laboratory testing conducted close to the
site of patient where care or treatment is provided
• Rapid test results, with the potential to generate a result quickly so that
appropriate treatment can be implemented, leading to improved clinical or
economic outcomes compared to laboratory testing.
