L3 Laboratory Diagnosis

Question 1

Role of laboratory diagnosis

Answer 1

Carry out tests in support of clinical diagnosis of infected disease etc

Question 2

Function

Answer 2

• Investigative advice
• Interpreting raw data
• Reporting of results
• Provision of treatment advice

Question 3

What are the types of specimen used?

Answer 3

Urine/ swab/ tissue/ blood/ sputum/ faeces

Question 4

Methods of microbiological diagnosis

Answer 4

Direct detection - look at sample down microscope
Culture - grow in lab and investigate it
Immunological test - not able to grow or see under microscope but able to see effects on body

Question 5

Microscopy

Answer 5

e.g. CSF examination in meningitis
Via gram method = detect presence of gram -ve diplococci in diagnosis (meningococcal meningitis)
Pros: Quick and provide info on how to treat

Question 6

Detect antigen

Answer 6

e.g. C.difficile (using glutamate dehydrogenase
Antigen-antibody complex is added to solution coupled to a dye (dye changes colour is correct complex is present)

• Lateral flow assay: cheap and rapid but not accurate
• 96-well format: manual or semi-automated
• Well based format: automated

Question 7

Detect nucleic acids

Answer 7

• Can be specific (directed against 1 target)

- Or amplify ‘unknown’ sequences using universal primers targeting 16s rRNA (matching to database)

Question 8

What are the uses and limitations of direct detection?

Answer 8

• Establish presence of microorganism at a particular site
• Allow use of appropriate empirical antimicrobial therapy
• Provides epidemiological info
• No info on susceptibility or typing
• Fast diagnostic method

Question 9

Culture requirements

Answer 9

Need culture medium (provides nutrients required for growth) and incubation (physical requirements for growth)

Question 10

Culture medium

Answer 10

• Non-selective: Growth of all microorganisms

- Selective: Inhibits growth of specific microorganisms (inhibitory components to certain microorganisms)

Question 11

What are indicator mediums used for?

Answer 11

Indicates certain properties of bacteria e.g. sugar fermentation

Question 12

MacConkey (indicator medium)

Answer 12

Contains sufficient water and nutrients to support bacterial growth
Dye turns red in presence of acid
Ferment lactose = red (acid) produced)
Can’t ferment lactose = yellow

Question 13

CLEO (indicator medium)

Answer 13

Lactose fermenter appears yellow

Question 14

Chromogenic agar

Answer 14

Often able to indicate more than 1 bacterial species

e.g. differentiate between E.Coli, Kiebsella spp. and Proteus spp. in urine

Question 15

Gram stain (Bacterial identification test)

Answer 15

Distinguish between bacterial cell wall
Ability to retain crystal violet
Gram +ve: purple
Gram -ve: pink (counterstain)

Question 16

Haemolysis (bacterial identification test)

Answer 16

Breakdown of blood cells
a = opaque
b = completely clear

Question 17

Oxidase test (bacterial identification test)

Answer 17

See whether have pseudomonas

oxidise to a purple colour

Question 18

Biochemical tests

Answer 18

• API strips contain lots of biochemical reactions all in 1 strip
• Produces profile to aid identification
• assign score to each positive test in a group of 3 tests
• Unique score from 0 to 7 is obtained
• Compare with database

Question 19

MALDI-TOF

Answer 19

• Mass spectrometer produces spectrum of bacterial proteins
• Compared to database of known spectra
• Cheap and fast
• Only as good as database

Question 20

Uses and limitations of bacterial identification tests

Answer 20

• Establishes presence of microorganism at particular site
• Allows use of empirical and targeted antimicrobial therapy
• Provide epidemiological and typing of information
• Usually slower than direct detection

Question 21

Susceptibility testing

Answer 21

Requires viable microorganisms (usually bacteria or fungi)

Question 22

Methods

Answer 22

• measure zone of inhibition
• commercial e-test
• breakpoint plates

Question 23

Uses and limitations of susceptibility testing

Answer 23

• inform decisions on targeted antimicrobial therapy
• initial treatment with ‘empirical’ therapy
• subsequent treatment is ‘targeted’

requires isolation of microorganism and antimicrobial susceptibility testing

Question 24

Immunological tests

Answer 24

IgM detection
Seroconversion = Change from -ve to +ve result from one test to subsequent test
Fourfold rise in titre = rise in concentration of antibody from one test to subsequent test

Question 25

Non-antibody immunological test

Answer 25

IFN-Y release assays in TB

Question 26

Uses and limitations

Answer 26

• Confirms exposure to specific microorganisms
• Provides epidemiological info
• Restricted to patients with detectable antibody response
• Is retrospective (too late to inform antimicrobial therapy decisions)