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Diagnosing Infections
To appreciate the principles underlying the strategies used to obtain a specific diagnosis of infection
Learning objective
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
How to Make a Specific Aetiological Diagnosis of Infection
• Demonstrate organism, component or product
• Isolate the micro-organism (“gold standard”)
• Demonstrate a serological response
Techniques to Demonstrate Organisms, Their Components and/or Products
• Microscopy
• Detecting specific components:
• Antigens
• Nucleic acids
• Other components
• Biological activity (e.g., toxin)
Demonstrating Infectious Agents
Microscopy:
• Unstained: wet, phase contrast, darkground
• Stained: Gram, Ziehl-Neelsen, etc.
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
Demonstrating Organisms and Components
Electron Microscopy:
50 nm
http://virology-online.com/viruses/Diarrhoea4.htm
Demonstrating Organisms and Components
• Microscopy
• Antigen detection
Latex Agglutination
1. Antigen in solution
2.
Latex particles coated with known antibody
Latex Agglutination (cont.)
3.
Cross-linking of latex particles
AO
label
known antibody
antigen of interest
Antigen Detection: Solid Phase Assay
solid phase
Direct Immunofluorescence of T. pallidum
Gilles & Dodds. Bacteriology Illustrated
Immunofluorescence Test for RSV
AO
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
http://www.leinco.com/immunohistochemistry
Demonstrating Specific Antigen in Tissues Using Immunohistochemistry
Substrate
Primary antibody (known specificity)
Labelled secondary antibody
Enzyme
Coloured product
Proteins
hepatocytes tumour
CAM5.2
S100
LCA
Use of Immunohistochemistry to Determine Cell Lineage
18
H&E
specific antibody to Ag of interest
label
solid phase
capture antibody
antigen of interest
Capture Assay for Antigen (in solution or not)
Demonstrating Infectious Agents and Components or Products
• Microscopy
• Antigen detection
• Nucleic acid detection
Demonstrating Infectious Agents and Products
Nucleic acid detection
• Hybridisation
• PCR
Principles of DNA Hybridisation
target -AGCACCGTCGGCCATCGTACAGGCCTACTAGCACTAGATGCTGACATT-
probe with label AGCCGGTAGCATGTCCGGA * *
probe with label AGCCGGTAGCATGTCCGGA * *
detect bound probe
• 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
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
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
Demonstrating Components or Products
• Antigen detection
• Nucleic acid detection
• Other components
Intact Cell Mass Spectrometry (IC-MS) (MALDI-TOF*)
• Uses whole bacteria
• Detects unique biomarkers or patterns
*Matrix-Assisted Laser Desorption Ionisation Time-of-Flight
How MALDI-TOF Works
Sandrin & Demirev. Microbe 2014; (1): 23
How to Make a Specific Aetiological Diagnosis of Infection
• Demonstrate organism, component or product
• Isolate causative agent
Isolating the Organism
Types of specimen:
• Sterile site
• Site with normal microbiota
• Sterile site abutting site with microbiota
Timing of specimen
Types of Culture Media
• Liquid (broth) vs solid (agar)
• Also: • selective • indicator • enriched • enrichment
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
How to Make a Specific Aetiological Diagnosis of Infection
• Demonstrate organism, component or product
• Isolate organism
• Demonstrate a serological response
Demonstrating an Antibody Response
Tube agglutination (Widal test)
AO
Solid Phase Assay
solid phase
patient’s sample
anti-human Ig
label
antigen (known)
* * *
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
Recent or Past Infection?
• Rising titre
• Specific IgM
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)
• Demonstrate organism, component or product
• Isolate organism
• Demonstrate a serological response
Summary
Obtaining a Specific Diagnosis of Infection