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Copyright regulations 1969

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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 microscopy 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


• 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


• 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



• 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



• Isolate organism


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)


• Isolate organism


Summary

Obtaining a Specific Diagnosis of Infection

copyright regulations 1969...bacteriology illustrated immunofluorescence test for rsv ao deposition...

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