First things first, what’s a PCR?
PCR stands for polymerase chain reaction (I find thinking of a parrot while humming the classic Diana Ross & Bee Gees song whenever seeing the word PCR jogs the memory). In a nutshell, it is a technique used to amplify a single copy, or a few copies, of a DNA segment resulting in the generation of thousands to millions of copies of a particular DNA sequence, from initially only a tiny amount.
Sounds very clever but how is that actually of use clinically to me?
If you are wanting to establish whether a particular organism is present on an animal a PCR can be used to amplify any DNA found belonging to that species so this can be detected. For example, if you are suspicious of, or want to rule out, fungal infections a PCR can inform you if dermatophytes are present in the submitted sample. Clinically, a positive result means at least part of the organism was present on the dog at the time of sampling. A huge advantage is the speed of a dermatophyte PCR result compared to fungal culture. The PCR also amplifies proportionally to what is present in the sample so is not affected by one species of fungi looking prolific simply because it grows well on the culture plate, it may therefore be more representative of what is on the animal’s skin.
The flowchart opposite shows how we see PCRs fitting in with more conventional tests to help rule out ectoparasites and fungal infections (to a complete work-up for a pruritic dog and cat click here).
Ah, but a fungal culture tells you about the species.
This is the clever bit! After dermatophyte detection by PCR, DNA sequencing, the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) within a DNA molecule, can then be used to identify the species of fungi that has been detected.
So why include fungal culture at all then?
If only life was so simple. One limitation of PCR is it’s not fussed if the DNA is dead or alive. Therefore, where it wins for detecting fungal infection, culture still is the best option to ensure successful resolution after appropriate treatment.
What about Sarcoptes, how does serology fit in with this?
Sarcoptes mites are notoriously pesky wee monkeys to detect on cytology with estimates of only 50% of true positives being detected on skin scrapes. While it is still absolutely worth going on a Scabies hunt it makes sense to use another technique that can inform you of recent exposure to the mite. This is especially true if you are trying to rule out these incredibly itchy pests in a work-up for atopic dermatitis before moving onto the next step.
Why bother with Sarcoptes PCR then?
Serological testing is looking at the IgG response which may take several weeks to decline after successful treatment. Sarcoptes serology is ideal when screening to see if there has been exposure to Sarcoptes mites but the PCR has the advantage for monitoring as you only get a positive if the DNA (so the mite) is still present.
But won’t the Sarcoptes PCR pick up dead Sarcoptes?
The ‘wanted dead or alive issue’ is not a problem as, unlike dermatophytes, Sarcoptes mites generally fall off when they’ve shuffled off this mortal coil.
What about the sensitivity and specificity of all these tests?
|Sarcoptes IgG serology||92.1%||94.6%|
*PCRs quoted figures are for the accuracy of detection of the organism from the submitted sample, therefore good sampling technique is paramount to optimise the performance of the test.
What can I do to get good samples?
We have guidance on sampling techniques (including a video guide) at www.avactaanimalhealth.com/samples.
Thinking about the predilection site of the organism you are trying to detect is also important. There are some excellent diagrams (see below for one example) showing the distribution of skin lesions associated with various infections/infestations in a paper by Hensel et al. from 2015, providing guidance on where to sample from.
Diagram showing the distribution of skin lesions for Sarcoptic mange:
Hensel, P., Santoro, D., Favrot, C., Hill, P. & Griffin, C. (2015). Canine atopic dermatitis: detailed guidelines for diagnosis and allergen identification. BMC Veterinary Research 11:196, DOI 10.1186/s12917-015-0515-5
That’s a lot of information to take in…
Hopefully this summary table, alongside the flowchart at the start of this article will help ?. To see how these tests fit into the complete work-up for a pruritic dog and cat follow this link www.avactaanimalhealth.com/home.
|Sarcoptes IgG Serology||Initial detection||Great to pick up exposure (rule out)||Canine||Serum|
|Sarcoptes PCR||Monitoring after treatment||IgG (serology) can remain elevated after treatment||Canine||Superficial skin scrapes|
|Dermatophyte PCR||Initial detection||Faster than culture||Canine, feline & others||Hair plucks, scale/crust, toothbrush sample|
|Dermatophyte sequencing||Identifying species (if + PCR)||Determine if species clinically relevant||Canine, feline & others||(already received for PCR)|
|Dermatophyte culture||Monitoring after treatment||Dermatophyte PCR may be + even when dead||Canine, feline & others||Hair plucks, toothbrush sample|
For full details on sample submission requirements, if you have any questions or if you would like any further information, please have a look on our website or contact our Customers Services on 0800 3 047 047.
Written by Johanna Gourlay– Senior Veterinary Technical Manager
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