copyright regulations 1969...bacteriology illustrated immunofluorescence test for rsv ao deposition...
TRANSCRIPT
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COMMONWEALTH OF AUSTRALIA
Copyright regulations 1969
Warning This material has been reproduced and communicated to you by or on behalf of the University of Melbourne pursuant to part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the act. Any further copying or communication of this material by you may be the subject of copyright protection under the act.
Do not remove this notice
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Diagnosing Infections
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To appreciate the principles underlying the strategies used to obtain a specific diagnosis of infection
Learning objective
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Diagnosing Infections
1. Make a clinical diagnosis based on: history and examination
2. Confirm with special investigations: imaging, lab tests, etc.
3. Don’t request tests blindly
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How to Make a Specific Aetiological Diagnosis of Infection
• Demonstrate organism, component or product
• Isolate the micro-organism (“gold standard”)
• Demonstrate a serological response
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Techniques to Demonstrate Organisms, Their Components and/or Products
• Microscopy
• Detecting specific components:
• Antigens
• Nucleic acids
• Other components
• Biological activity (e.g., toxin)
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Demonstrating Infectious Agents
Microscopy:
• Unstained: wet, phase contrast, darkground
• Stained: Gram, Ziehl-Neelsen, etc.
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Ziehl-Neelsen stain of sputum showing
acid-fast bacilli
Examples of the Usefulness of Light Microscopy
Gram stain of urethral pus showing
gonococci
Phase contrast micros- copy of faeces showing
trophozooites of Giardia lamblia
AO AO AO
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Demonstrating Organisms and Components
Electron Microscopy:
50 nm
http://virology-online.com/viruses/Diarrhoea4.htm
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Demonstrating Organisms and Components
• Microscopy
• Antigen detection
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Latex Agglutination
1. Antigen in solution
2.
Latex particles coated with known antibody
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Latex Agglutination (cont.)
3.
Cross-linking of latex particles
AO
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label
known antibody
antigen of interest
Antigen Detection: Solid Phase Assay
solid phase
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Direct Immunofluorescence of T. pallidum
Gilles & Dodds. Bacteriology Illustrated
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Immunofluorescence Test for RSV
AO
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Deposition of IgA (yellow-green staining) in the glomerulus, detected by immunofluorescence. Patient has IgA nephropathy
Also Used in Anatomical Pathology
For example: To detect:
• immunoglobulins in kidney biopsies or
• immune complexes in skin biopsies
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http://www.leinco.com/immunohistochemistry
Demonstrating Specific Antigen in Tissues Using Immunohistochemistry
Substrate
Primary antibody (known specificity)
Labelled secondary antibody
Enzyme
Coloured product
Proteins
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hepatocytes tumour
CAM5.2
S100
LCA
Use of Immunohistochemistry to Determine Cell Lineage
18
H&E
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specific antibody to Ag of interest
label
solid phase
capture antibody
antigen of interest
Capture Assay for Antigen (in solution or not)
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Demonstrating Infectious Agents and Components or Products
• Microscopy
• Antigen detection
• Nucleic acid detection
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Demonstrating Infectious Agents and Products
Nucleic acid detection
• Hybridisation
• PCR
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Principles of DNA Hybridisation
target -AGCACCGTCGGCCATCGTACAGGCCTACTAGCACTAGATGCTGACATT-
probe with label AGCCGGTAGCATGTCCGGA * *
probe with label AGCCGGTAGCATGTCCGGA * *
detect bound probe
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• Detect DNA or RNA e.g. • infectious agents in tissues • overexpression of HER2 in breast cancer
http://www.her2sish.com/main.php
In Situ Hybridisation in Tissue Sections
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Principles of PCR
• Target DNA is “melted” to give single strands
• Primer pair binds to homologous sequences in target (one to each strand)
• DNA polymerase is used to synthesise DNA extending from bound primer and incorporating free nucleotides from the solution
• This yields two new strands of DNA; each complementary to & bound to one strand of the target
• This process is repeated 30 to 40 times
• Product detected and identified
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Using PCR to Identify the Causative Agent of Bacterial Meningitis in Children
• need to confirm identity (e.g., by RFLP analysis, hybridisation, sequencing)
513 bp Haemophilus influenzae
1492R HI
679 bp Neisseria meningitidis
1492R NM
265 bp Streptococcus pneumoniae
1492R STREP
PCR primers used (based on 16S rRNA)
265 bp
513 bp 679 bp
1 2 3
Isolation of PCR Products
AO
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Demonstrating Components or Products
• Antigen detection
• Nucleic acid detection
• Other components
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Intact Cell Mass Spectrometry (IC-MS) (MALDI-TOF*)
• Uses whole bacteria
• Detects unique biomarkers or patterns
*Matrix-Assisted Laser Desorption Ionisation Time-of-Flight
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How MALDI-TOF Works
Sandrin & Demirev. Microbe 2014; (1): 23
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How to Make a Specific Aetiological Diagnosis of Infection
• Demonstrate organism, component or product
• Isolate causative agent
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Isolating the Organism
Types of specimen:
• Sterile site
• Site with normal microbiota
• Sterile site abutting site with microbiota
Timing of specimen
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Types of Culture Media
• Liquid (broth) vs solid (agar)
• Also: • selective • indicator • enriched • enrichment
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Confirmatory and Further Tests
• Confirm identity using biochemical tests,
MALDI-TOF or DNA/RNA analysis
• Determine subtype, e.g.
• serotype, pathotype, etc.
• Determine antimicrobial susceptibility
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How to Make a Specific Aetiological Diagnosis of Infection
• Demonstrate organism, component or product
• Isolate organism
• Demonstrate a serological response
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Demonstrating an Antibody Response
Tube agglutination (Widal test)
AO
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Solid Phase Assay
solid phase
patient’s sample
anti-human Ig
label
antigen (known)
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* * *
Protein Gel
2. Transfer
3. Add patient’s serum
4. Add 2nd Ab to reveal bound Ab
lmmunoblot (Western blot)
1. Separate proteins by electrophoresis
AO
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Recent or Past Infection?
• Rising titre
• Specific IgM
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Assays for Specific IgM Antibodies
solid phase
patient’s sample
anti-human IgM
label
antigen (known)
patient’s sample
specific Ab (known)
label
solid phase
anti-IgM
antigen (known)
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• Demonstrate organism, component or product
• Isolate organism
• Demonstrate a serological response
Summary
Obtaining a Specific Diagnosis of Infection