MB 5 - Diagnostic Techniques

Question 1

Learning Outcomes (for general perusal)

Answer 1

Be able to describe with examples

• Direct and indirect diagnostic tests
• Microscopy and specifically the gram stain
• The principles of culture of microbes
• The basics of antibody detection methods for diagnosis of infection
• The basics of molecular detection of infectious agents
• The approach to taking blood cultures

Question 2

What can microbiology be used for?

Answer 2

• Treatment
• Prognosis
• Infection control
• Epidemiology

Question 3

Give examples of some infectious diseases

Answer 3

• Mumps
• Chickenpox
• SARS
• Tetanus
• Diphtheria

Question 4

Microbiological tests can be…

Answer 4

• Diagnostic - What infectious agent is there? Is this significant?
• Theronostic (to direct therapy) - more about the bug
  
  • Viral load
  • serial testing
  • Sensitivity/ resistance testing
• Immunity Testing - Has the person responded to a vaccine? Does the exposed person need prophylaxis?

Question 5

1. Outline Direct testing
2. Outline Indirect testing

Answer 5

1. See a germ, Grow a germ, Find a bit of germ (antigen), Find a bit of germ nucleic acid
2. Find evidence of a specific immune response to a germ (lymphocytes, antibodies)

Question 6

Give an example of direct testing

Answer 6

See a germ - Microscopy e.g Gram Stain (Gram negative diplococci In PMN in CSF =Meningococcal meningitis)

Grow a germ - eg. Staphylococcus aureus, Allows antibiotic sensitivities to be determined

Find a bit of germ (antigen) - Legionella urinary antigen

Find a bit of germ nucleic acid - PCR

Question 7

Give an example of indirect testing

Answer 7

Finding evidence of a specific immune response to a germ - in practice this usually means serology

• Ab Assay Examples
  
  • CFT (complement fixation test)
  • ELISA (enz-linked immunosobent assay) / EIA (enz immunoassay) - same thing
  • Indirect immunofluorescence (IIF)
• Measurements
  
  • IgG / IgM (short lived = recent infection) / IgA Total Antibody or Positive, Negative, Equivocal or Quantity in Units
  • Rising levels (serial sampling)
  • IgG Avidity (estimation of how recent)
  • Immunoblots (reactivity to xple Antigens from a bug)

Question 8

Diagnostic Process

What is the role of the microbiology lab?

Answer 8

• Clinical history and examination
• Differential diagnosis
• Investigations

–Laboratory investigations

•Microbiology investigations

–Serology

–Direct detection

Question 9

• *Clinical Diagnosis example**
• Meningococcal meningitis*

1. What would the patient present with?
2. What simple clinical test can be done?
3. What microorganism would be seen microscopically?
4. Describe it’s appearance
5. What microbiological tests are carried out?

Answer 9

1. Headache, fever, vomiting,drowsiness, Haemorrhagic rash, neck stiffness. Infants = poor feeding, bulging fontanelle
2. Tumbler Test Positive in meningococcal infection
3. Neisseria meningitidis
4. Gram negative diplococci
5. Microscopy

PCR

Culture

Question 10

What determines if a good specimen is taken?

Answer 10

–site

–technique

–timing

–transport time

–storage conditions

–transport medium

Question 11

Give examples of some specimens that can be taken?

Answer 11

–Throat swab

–Sputum

–Blood

–Faeces

–CSF

–Genital swab

–Biopsy

Question 12

Give the different types of blood samples

Answer 12

• Clotted blood
  
  • Serum e.g. serology
• EDTA blood
  
  • Plasma / WBCs / Whole Blood e.g. PCR
• Blood culture

EDTA = Ethylenediaminetetraacetic acid. Anticoagulant, prevents haemolysis,

Question 13

How will a diagnosis come about in a

1. Patient
2. Specimen

Answer 13

1. Clinical Diagnosis. Haematology, Biochemistry, Non-microbiological investigations eg. Radiology
2. Correct specimen, taken correctly, labelled and packaged correctly, appropriate transport and storage of specimen

Question 14

1. What is sensitivity?
2. What is specificity?
3. What is positive predictive value?
4. What is negative predictive value?

Answer 14

1. Measure of the lack of false negatives
2. Measure of the lack of false positives
3. TP/TP+FP
4. TN/TN+FN

Question 15

MICROSCOPY

1. What can be seen with an electron microscope?
2. What can be done with a light microscope?

Answer 15

1. Herpes Virus, Paramyxovirus
2. Staining e.g Gram Stain

Question 16

MICROSCOPY

Outline the process of Gram staining

Answer 16

1. Crystal Violet - Everything stains
2. Gram’s Iodine - Stain differently fixed
3. Decolorise (Acetone) - Gram-negatives destain
4. Counterstain with Methyl Red - Gram-positives appear purple, Gram-negatives appear pink

Question 17

Microscopy

Name some different shapes and gram reactions

Answer 17

• Gram positive cocci
• gram-positive rods
• gram-negative cocci
• gram-negative rods

Question 18

What is the appearance of

1. Neisseria meningitidis in CSF
2. Staphylococcus aureus and E. coli - surgical wound

Answer 18

1. Gram negative diplococci (intracellular)
2. Gram positive cocci & gram negative rods

Question 19

Microscopy

​Describe the appearance of

1. Haemophilus influenzae
2. Streptococcus pyogenes

Answer 19

1. Pleomorphic
2. Gram-positive connected in long chains

Question 20

What can the Ziehl-Neelsen (ZN) stain be used for?

Answer 20

Used for the bacteria that cause TB

Sputum

Old technique

Question 21

‘Grow a germ’

1. What is a Bacteriological culture?
2. What types of atmospheres can the culture be incubated in?
3. Give an example of colony appearance

Answer 21

1. Clinical specimen inoculated onto various media (selective, enrichment). Appearance of colonies suggests species present. Further tests to clarify
2. Anaerobic, Aerobic, CO₂-rich
3. Haemolysis
   
   1. Beta-complete, Clear zone, B haemolysis, blood has been lysed = Streptococcus pyogenes
   2. Alpha-incomplete, Green zone, blood hasn’t been lysed, made leaky = Streptococcus pneumoniae

Question 22

Antimicrobial Sensitivities

Outline these

Answer 22

• Filter paper disc
• Impregnated with antibiotic
• Zone of inhibition

If it can grow right up to the disc = resistant

Question 23

What is a viral culture conducted in?

Giving this test what characterisitics?

Answer 23

Living cells

Slow and sensitive

Question 24

Molecular Tests

What do these involve?

Answer 24

• Detection of nucleic acid
• Quantitative or Qualitative
• Amplification methods e.g. PCR

Question 25

**Molecular Tests** **Quantitative** 1. Give an example

Answer 25

1. How much eg. HIV load (Viral RNA in blood) 1. Can be done before and during treatment

Question 26

**Polymerase Chain Reaction (PCR)** ## Footnote Outline this process What can be a limitation, but at the same time a benefit?

Answer 26

* Specific primers - need to know what you're looking for. BUT lends extra specificity * 20 nucleotides * Melt * Anneal primers * Extend new DNA * 25- 35 cycles

Question 27

1. Outline **Realtime PCR** 2. What can carry out Single Test Realtime?

Answer 27

1. PCR is quatifiable – can work out how long it takes to become positive Good at saying how much there is in a sample Going up, going down, staying the same 2. Xpert MTB/RIF - a test to detect tuberculosis and rifampicin resistance directly from sputum in less than 2 hours

Question 28

What does Molecular Diagnosis of Rifampicin Resistance (TB) involve?

Answer 28

DNA Extraction PCR Detection Valid Results

Question 29

**Indirect Testing** Give examples of methods of **serology**

Answer 29

* **Complement fixation test** * Ag-Ab complex fixes complement * **Enzyme immunoassays (ELISA)** * colour change * **Immunofluorescence** * labelled antibody

Question 30

**Serology** What does the Complement fixation test (CFT) involve?

Answer 30

* Positive result = no RBC lysis * complement 'fixed', can't lyse * complement attached to patient antibody which is attached to antigen * Negative Result = RBC lysis * When complement is added, cells burst, no antibody to attach to If have antibody, will create antigen antibody complex, complement will stick to. Complement sucked out. RBC doesn’t burst

Question 31

**Serology** What do Enzyme immunoassays EIA or ELISA involve?

Answer 31

Enzyme mediated colour change An enzyme - labelled anti-human antibody is added This will attach to human antibody which is attached to an antigen (eg.HIV) Enzyme will change from colourless-coloured

Question 32

**Serology** What does **Immunofluorescence** serology involve?

Answer 32

Mouse anti-human Ig with a fluorescent label added Will attach to patient's antibody attached to antigen eg. HSV Can see if there is a patient antibody there or not Can do serial diluations of the blood see where it goes from positive to negative.

Question 33

**Examples of Microbiology Testing** *_Chicken Pox_* 1. What is the earliest diagnostic microbiological test? 2. When will blood tests be effective?

Answer 33

1. PCR (from day 14) 2. IgM (day 18) and IgG (day 20) - blood tests will give negative results earlier in the course

Question 34

**Examples of Microbiology Testing** *_Staphylococci & Streptococci_* 1. What is their appearance on gram stain? 2. How will Staph.aureus look? 3. How will Strep.pyogenes look?

Answer 34

1. Gram positive cocci 2. In clusters, catalase-positive 3. In chains, catalase-negative

Question 35

**Examples of Microbiology Testing** *_Streptococcal pharyngitis_* What does diagnosis involve?

Answer 35

* Throat swab * Plate out (Blood agar) * Beta-haemolysis (complete haemolysis) * latex agglutination typing test (Lancefield grouping of capsular antigen) * (Result is Group A)

Question 36

**Examples of Microbiology Testing** *_Dermatophyte Infections_* ​How is a diagnosis made?

Answer 36

* direct microscopic examination of scales dissolved in KOH * by culture of scrapings of lesions

Question 37

**Examples of Micriobiology Testing** *_Bronchiolitis_* 1. What microorganism is likely to be present? 2. What will the patient present with? 3. How is it diagnosed? 4. What specimen is used?

Answer 37

1. RSV - Respiratory syncytial virus 2. febrile wheeze can progress to respiratory obstruction or exhaustion, requiring hospitalisation 3. **Immunofluoresence** or **PCR** 4. **nasopharyngeal aspirate**

Question 38

Outline the Identification and typing of organisms

Answer 38

* **Antigenic type** * e.g. Serotype * **Genotype** * e.g. RFLP (Restriction Fragment Length Polymorphism), Sequence * Others * biotype eg API biochemical tests * antibiotic resistance pattern

Question 39

**Examples of Microbiology Testing** *_Salmonella_* What does it involve?

Answer 39

* **Biochemical tests** * Commercial kits commonly used, e.g. API20 * **Antigenic tests** * Sero-typing agglutination test

Question 40

**Blood Cultures** Outline the principles

Answer 40

* Sensitivity increases with quantity of blood * Take 10-20 ml per set * **THREE** blood culture sets usually taken except in suspected endocarditis when up to SIX are taken. * Samples can be taken through lines if line infection suspected

Question 41

**Blood Cultures** 1. How should they be stored?

Answer 41

* After blood cultures have been taken they are placed in an incubator immediately for maximum speed & sensitivity * never put blood cultures in the fridge!!! * Incubate at 37°C for up to 5 days * longer in endocarditis, PUO (pyrexia of unknown origin)