32 Diagnosis of infection and assessment of host defense mechanisms Flashcards
What is involved in pre-analytical stage of laboratory practice?
Appropriate selection of tests
Proper filling in request forms
Collection/ transport specimens
Check patient details on sample/ paperwork
What are routes of potential infection for lab staff in the lab?
Inhalation - aerosol
Ingestion - eating/ drinking in lab
Skin penetration/ uncovered wound
34 year old with two days fever, abdo pain, diarrhoea with blood, vomiting. Other people have similar symptoms.
What tests should be performed?
MC&S - like campylobacter or salmonella
No need viral PCR - not norovirus
No need oca/ cysts/ parasites
Asked to introduce new POC HIV test to A&E.
Which do you pick?
Sens 22% Spef 100%
Sens 50% Spef 50%
Sens 98% Spef 22%
Sens 98% Spef 22%
Sensitivity correctly identifies those with virus.
Specificity correctly identifies those without virus.
Tests with high sensitivity are used to rule in disease.
Tests with high specificity are used to rule out disease.
Screening test should be highly sensitive, and confirmatory test should be highly specific.
This way we will pick up 98% of all HIV coming to A&E. There will be some false positives, but this will be excluded on confirmatory test. Better to over-diagnose, than miss a case of HIV
What is positive predictive value?
What is negative predictive value?
PPV - reflects probability that HIV is present when test is positive
NPV - reflects probability that HIV is absent when test is negative
PPV is influeced by sensitivity/ specificity, and also the prevalence of a disease in a population
What are main techniques when processing bacteriology specimens?
Microscopy - use Neubauer champer to count WBC, gram stain
Culture - streak out on agar plate to obtain single colony
Sensitivity testing
MALDI-TOF - mass spectrometry
What are names of different types of agar used to culture bacteria?
Samples from faeces require selective agar, so that commensals do not grow
Blood agar
Chocolate agar
MacConkey agar
Sorbitol MacConkey agar
Xylose lysine deoxycholate (XLD) agar
Chromogenic MRSA
Camyplobacter agar
Neomycin fastidious anaerobe agar
TCBS agar
Columbia CNA agar
What organisms are these culture media used for?
Blood agar
Chocolate agar
MacConkey agar
Blood agar - most bacteria. Look for haemolysis if streptococci
Chocolate agar - fastidious H influenzae/ N meningitidis
MacConkey agar - to separate lactose fermenters
- lactose fermenter - E. coli/ klebsiella
- non-lactose fermenter - Salmonella/ shigella
What organisms are these culture media used for?
Sorbitol MacConkey agar
Xylose lysine deoxycholate (XLD) agar
Sorbitol MacConkey agar - E.coli O157
Xylose lysine deoxycholate (XLD) agar - salmonella/ shigella
What organisms are these culture media used for?
Chromogenic MRSA
Camyplobacter agar
Neomycin fastidious anaerobe agar
Chromogenic MRSA - MRSA
Camyplobacter agar - campylobacter. Seagull shaped gram-negative bacteria
Neomycin fastidious anaerobe agar - clostridium perfringens
What organisms are these culture media used for?
TCBS agar
Columbia CNA agar
TCBS agar - vibrio cholerae
Columbia CNA agar - gram positive bacteria e.g staphylococci, streptococci, enterococci
What is catalase test, and what is it used for?
Tests for presence of enzyme catalase, which is present in staphylococcal species (not streptococci)
Add hydrogen peroxide to colonies, and add cover slip. If catalase present, will convert hydrogen peroxide to water and oxygen and release bubbles
What is oxidase test, and what is it used for?
Used to test for pseudomonas species. Helps differentiate from other gram negative rods e.g E.coli, klebsiella
Add oxidase reagent to colony. If cyctochrome c oxidase enzyme present, will change to purple
What is urease test, and what is it used for?
Urea in culture medium is broken down by organisms possessing urease, into ammonia and carbon dioxide. Colour change from yellow to red-orange
Species with urease - proteus providencia klebsiella morganella ureplasma helicobacter pylori Nocardia cryptococcus
What are steps of gram staining?
- Heat-fixed cells are stained with crystal violet stain, which colours the cells purple
- Iodine solution is added, which forms a complex with the crystal violet stain and traps it within the cell
- Cells are washed with ethanol solution (acetone), which decolorises Gram-negative bacteria (but not Gram-positive)
- Gram-negative cells are visualised by applying safranin – which is a pink-coloured counterstain
Gram staining works as a result of the different effects of ethanol on the cell wall of the bacteria:
Gram-positive bacteria have a thick peptidoglycan layer which is dehydrated by ethanol, trapping the crystal violet stain - appearing purple.
Gram-negative bacteria have an outer lipid membrane which is dissolved by ethanol, releasing the crystal violet stain
Both cell types retain the counterstain, but the pink colour cannot be distinguished in the purple-stained Gram-positive cells
How are mycobacterium stained?
They have large lipid coat, so do not take up gram stain
Auramine stain is more sensitive/ less specific. So can use with microscopy as screening test
Ziehl Neelsen stain used as confirmatory test
How are cryptosporidium dtected?
Auramine stain
Examine under UV fluorescence microscope
What are tests to detect antimicrobial sensitivity?
Disk diffusion - compare zone of growth inhibiton around colony
E-strip - gradient to determine which concentration inhibits growth
If culture results are negative, what further molecular tests can be performed?
16S PCR is used to detect presence of organism using real-time PCR
Microorganisms have their own unique 16s rRNA gene