Users Guide to Laboratory Diagnostics

Why is it necessary to identify infectious diseases?

• knowledge of likely spread

• epidemiology

• choice of treatment

How can we diagnose infection?

• grow the agent

• look for immune response to the agent

• look for DNA/RNA of the agent

• look for pathological signs

What should you consider when growing a culture?

• aerobic/anaerobic

• can it cause disease or is it a contaminant?

• how quickly does it grow?

• can I grow it at all?

What are the pros of growing pathogen?

• know you have a live organism

• positive identification

• no prediction necessary

• can test it for antibiotic resistance

What are the cons of growing pathogen?

• false negative are common for some disease

• slow

What does this image show?

Dipstick tests

What does this image show?

ELISA

What does this image show?

microscopic agglutination test

How can you look for an immune response?

• dipstick tests

• ELISA

• microscopic agglutination test (MAT)

What can you use a microscopic agglutination for?

leptospirosis

How do you look for an immune response?

• look for antigen circulating in blood - use antibody to capture this

• can look for antibody - IgG or IgM using antigen to capture this

Antigens and antibodies binding makes complexes that are either:

• MAT - visible

• detected by using a second antibody against the antibody or antigen once it has been captured

What are ELISA tests?

coated plates used to analyse fluid samples

What are ELISA plates coated with?

• known antigen

• antibody

Why can you get false positives from ELISAs?

• vaccination

• maternal transfer

• historic infection

What do ELISAs detect?

• antibody level in samples (if coated with known antigen)

• antigen (if coated with antibody)

What do you look for in an ELISA when coated with antibody to detect circulating antigen?

unique parts of viruses, fungi, bacteria

What causes a colour change in a substrate in ELISA?

second antibody attached to an enzyme

What are the pros of dipsticks?

• fast

• can be done while patient is in the room

What are the cons of dipsticks?

• you need to suspect something to test for it

• cross reactivity can be a problem

What are the pros of looking for an immune response to diagnose?

• fast

• indicates recent (IgM) or historical (IgG) exposure OR presence - antigen

What are the cons of looking for an immune response to diagnose?

• you have to predict what you are looking for

• you can miss new infections - antigen variation confuses the test

How can you look at DNA to diagnose?

PCR

What are the pros of PCR?

• sensitive

• good for viruses

• relatively fast

What are the cons of PCR?

• need to predict what you are looking for

• doesn’t indicate amount (so has to be used for obligate pathogens)

What is real time PCR?

• like PCR but based on the incorporation of dye into the growing amount of PCR product and monitoring this

• or based on the release of fluorescent tags from the DNA products as they are made

What are the pros of real time PCR?

• fast

• specific

• quantifies the agents so can be used for infections that are opportunistic

What are the cons of real time PCR?

• need to know what you are looking for (panels)

• internal controls needed - do you even have DNA

What test is quickest?

antibody

What test is most specific?

real time PCR

Gram?

negative - pink

What sort of atmosphere does this like growing in?

anaerobic - spore former

Are hopanoids, peptidoglycan layers or lipopolysaccharide gram positive?

peptidoglycan layers

Are hopanoids, peptidoglycan layers or lipopolysaccharide gram negative?

lipopolysaccharide (LPS)

Are hopanoids, peptidoglycan layers or lipopolysaccharide gram positive and gram negative?

hopanoids

Name somewhere a biofilm can form in a patient

• heart valve

• urinary catheter

Under what conditions can a facultative anaerobe grow?

• atmospheric oxygen

• no oxygen

• low oxygen

• all of them

ALL - facultative means it’s adaptable

Your feet smell of cheese because bacteria there produce:

• ethanol

• methane

• lactic acid

• formic acid

• butyric acid

lactic acid

How do the bacteria on your feet produce lactic acid?

fermentation

What are methane, hydrogen and sulphide produced by?

• fermentation

• aerobic respiration

• anaerobic respiration

anaerobic respiration

What does an ELISA measure?

• DNA or RNA

• antigen or antibody

• prokaryotic DNA or antigen

• IgG or IgM

antigen or antibody

What is this?

snap/dipstick test

What does this snap/dipstick test measure?

lateral flow device

If you use this and got a positive result for IgM against SARS-CoV2 in a 3yr old cat, are they likely to have the infection?

IgM indicates current infection, so unless it is vaccinated, YES

If you use this and got a positive result for IgM against SARS-CoV2 in a 3 week kitten, are they likely to have the infection?

not clear, could be maternal transfer, not a good test

If it was IgG and positive, would this be a better test for disease? why?

NO IgG indicates historical infection

If you do a PCR for SARS-CoV2 and it’s positive does this indicate infection?

better than dipstick - tells you that nucleic acid and so live virus is there

If you do a swab from your nose and it’s positive for Staphylococcus aureus, should you visit your GP?

NO - staph is a normal commensal here

If you do a swab from a cut on your leg and it’s positive for Staphylococcus aureus, should you visit your GP?

probably not - you don’t know whether there’s lots of bacteria or just a few - it’s a qualitative test - look for signs of infection too