Lecture 18 - Diagnosing Infections Flashcards by Jack Cuthbertson

Stages of diagnosis
1)
2)

1) Clinical diagnosis, based on history, examination

2) Lab diagnosis

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How to make a specific aetiological diagnosis of infection
1)
2)
3)

1) Demonstrate organism, component or product
2) Isolate organism
3) Demonstrate serological response

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How are lab tests used?

Often use more than one, as a single positive result doesn’t necessarily prove causation
EG: Isolating S. typhae in someone’s faeces doesn’t prove Salmonella causing typhoid fever, as some people carry S. typhae asymptomatically

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What are Ziehl-Neesen stains good for diagnosing?

Open pulmonary TB infections

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Microscopy techniques for diagnosis
1)
2)
3)

1) Unstained: wet, phase contrast, darkground
2) Stained: Gram, Ziehl-Neesen
3) Electron microscopy

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Organism that phase-contrast microscopy can detect

Giardia lamblia

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Organism that Gram staining can detect

Neisseria gonorrhoeae

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Organism that Ziehl-Neesen staining can detect

Mycobacterium tuberculosis (or acid-fast bacteria)

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How can Giardia lamblia be imaged?

Phase-contrast microscopy

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How can Neisseria gonorrhoeae be imaged?

Gram staining

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Methods of antigen detection
1)
2)
3)

1) Latex agglutination assay
2) Solid-phase assay
3) Capture assay for antigen

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Latex agglutination method
1)
2)
3)

1) Antigen sample is in solution
2) Latex particle with antibodies of known specificity are in solution
3) If antibodies can bind to antigen, then they cross-link, agglutinate

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Interpretation of latex agglutination results

Positive result = clear solution (agglutination of latex beads)
Negative result = cloudy solution

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Solid-phase assay technique
1)
2)
3)
4)

1) Antigen of interest is fixed on solid phase
2) Known antibody is washed over antigen of interest.
3) Antibody has a marker attached to it, which can be detected (EG: fluorescent probe)
4) If a positive result, probe can be detected on sample

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Example of where solid-phase assay is used

To test for T. pallidum

Uses a fluorescent probe (direct immunofluorescence)

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Capture assay for antigen technique
1)
2)
3)
4)

1) Capture antibody of known specificity is fixed to solid phase
2) Sample is washed over solid phase. Might include antigen being tested for,
3) Another antibody specific to the antigen of interest is washed over solid phase. Antibody has an attached label (radioactive, fluorescent, etc)
4) If antigen being tested for is present, then it will bind to solid phase antibody and be bound by liquid phase antibody with marker.

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When is capture assay for antigen used?

When a sample is messy, with lots of potential antigens

EG: faecal sample, testing for rotavirus, etc

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Example of a test for biological activity

Testing for toxin

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Toxin presence test
1)
2)

1) Culture of vero cells

2) Incubate vero cells with sample. If eg: verotoxin/shigatoxin is present, then cell death

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Toxin type test
1)
2)
3)

1) Incubate culture of vero cells, sample and antibodies against specific toxin (EG: verotoxin)
2) If antibodies are specific to toxin, then toxin is neutralised, cells are not killed.
3) Compare to control without antibodies

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Two types of test for nucleic acids

1) Hybridisation (older test)

2) PCR

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DNA hybridisation
1)
2)
3)
4)

1) Place sample, oligonucleotide probe of known sequence together.
2) Oligonucleotide probe is labelled (EG: radioactive)
3) Melt DNA into single strands, see if probe binds to complementary sequence
4) If probe has bound, can detect label in sample (EG: X-ray paper for radioactive probe)

Using PCR to determine causative agent of bacterial meningitis in children
1)
2)
3)
4)

1) PCR primers for 16S rRNA for haemophilus influenzae, streptococcus pneumoniae, neisseria meningitidis
2) Perform PCR
3) Isolate PCR products, run on a gel
4) Each of the three bacteria have different fragment lengths.

Issue with using PCR to determine causative agent of bacterial meningitis

Can’t prove causative agent, can only show that there is an RNA sequence present in sample that is of same length to rRNA of potentially-causative bacterium.

Must conduct other tests to confirm (EG: RFLP analysis, hybridisation, sequencing)

Main causative agents of bacterial meningitis in children 1) 2) 3)

1) Neisseria meningitidis 2) Streptococcus pneumoniae 3) Haemophilus influenzae (there is a vaccine, but it is expensive. Not used in poorer countries)

How does MALDI-TOF detect bacteria? 1) 2) 3)

1) Place pure sample of whole bacteria in spectrometer 2) Detects unique biomarkers of specific bacteria 3) Results are compared to global database of results

Advantages of MALDI-TOF

99.9% accurate identification | Only takes a few minutes to identify bacteria

Disadvantages of MALDI-TOF

Can't identify O or H group of bacteria. EG: Can say that sample is E. coli or not, but not O or H group of E coli (can't tell whether a pathogenic or non-pathogenic serotype of E coli is present)

Isolation of causative organism considerations 1) 2) 3)

1) Type of sample 2) When sample was taken 3) Which culture to use

Types of samples 1) 2) 3)

1) Sterile site 2) Site with normal microbiota 3) Sterile site abutting site with normal microbiota

Example of a sterile site

Blood, CSF

Example of a site with normal microbiota

Samples from GIT (EG: faecal)

Example of a sterile site abutting a site with normal microbiota

Urine sample passed through the urethra Lung sputum sample passed through upper respiratory tract

Why can it be difficult to diagnose urinary tract infections and lower respiratory tract infections?

Most common pathogens for LRTI, UTI are common microflora in the urethra and upper respiratory tract. These contaminate samples

``` Types of media 1) 2) 3) 4) ```

1) Selective 2) Indicator 3) Enriched 4) Enrichment

Selective media

Will only allow a particular type of bacteria to grow on it EG: MacConkey agar - contains bile salts, will kill respirartoy bacteria

Indicator media

Indicates presence of a bacterium with a particular phenotype EG: Blood agar, can show haemolysis

Bacterium that causes beta-haemolysis

Beta-haemolytic streptococcus

Enriched media

Has nutrients to make it easier for bacteria to grow | EG: Chocolate agar

Enrichment media

Makes it easier for a particular type of bacteria to grow EG: Selenite broth: Selenite doesn't inhibit Salmonella growth Virbio cholerae likes alkaline environments, other bacteria don't (~pH 8.5) (TCBS agar)

What is MacConkey agar?

Selective and indicator

What is blood agar?

Enriched and indicator

Easy way to conduct biochemical tests

API 20E Multistrip

Confirmatory tests

Confirm using biochemical tests or DNA analysis

Further tests after identification of organism 1) 2)

1) Identify subtype (phage type, serogroup, pathotype) | 2) Identify antimicrobial susceptibility

Demonstration of a serological response 1) 2)

1) Take patient serum sample | 2) Mix with antigen of known specificity

Difference between antigen test and serological test

In antigen test, antigen is unknown, antibody is known In serological test, antibody is unknown, antigen is known

``` Serum test for antibodies against S. typhi 1) 2) 3) 4) 5) ```

1) Make doubling dilutions of patient serum 2) Add heat-killed S typhi, incubate for 24 hours 3) If there are no serum antibodies, sample is cloudy, as cells are in suspension 4) If there are serum antibodies, cells agglutinate, sink to the bottom. Sample is clear 5) Titre is determined as the minimum concentration of antibody that crosslinks sample

What is a better version of the serum test?

Solid-phase assay

``` Solid phase assay for antibody 1) 2) 3) 4) ```

1) Known antigen is immobilised on solid phase 2) Wash with patient's sample 3) Wash with anti-human antibody with a probe 4) Test for presence/absence of probe

Conditions for execution of solid phase assay for antibody 1) 2) 3)

1) Antigen must be in excess 2) Patient sample must be limiting factor (diluted) 3) Therefore, the more antibody present in the sample, the darker a positive result will be

``` Western blot 1) 2) 3) 4) ```

1) Separate known proteins using gel electrophoresis 2) Transfer electrophoresed proteins to a piece of paper 3) Add patient serum sample to paper 4) Add second antibody (anti-human antibody) to sample, if it binds, positive result

Use of western blotting

HIV test | Tests for presence of anti-HIV antibodies

How is an HIV test carried out? 1) 2) 3)

1) Run a western blot of several HIV antigens 2) If patient serum antibodies bind to more than one antigen, true positive result 3) If patient serum antibodies only bind one protein, retest after a few weeks. Could be detecting early HIV infection, or false positive.