1 www.catvirus.com Feline Infectious Disease Masterclass Dr Diane D. Addie __________________’s workbook page SATURDAY Diagnosis of effusive FIP using an algorithm lecture 2 Dr Soma’s probability of wet FIP table 3 Case history: Mirabelle 4 Case history: Jess 8 Workshop 1: effusive FIP diagnosis 11 Case history: did this cat get FIP when you spayed her? 11 Case history: does Lisa have FIP? 12 Case history: does Oliver have FIP? 17 SUNDAY Non-effusive FIP lecture 18 Workshop 2: non-effusive FIP diagnosis Case history: does Zena have FIP? 23 Case history: does Buffy have FIP? 24 Case history: does Tommy have FIP? 25 Case history: does Basil have FIP? 29 Recommended laboratories and contacts 30 References 30 APPENDICES AND SPARE WORKSHEETS Catvirus.com FIP diagnosis worksheet template 33 FIP diagnostic flowchart Step 1 for the cat’s guardian to complete 34 Treatment protocol for effusive FIP 35 Treatment protocol for non-effusive FIP 36 Effusive FIP diagnosis algorithm 37 Non-effusive FIP diagnosis flowchart 38 FCoV GIT diagnosis flowchart 39 Spare flowcharts for use in the workshop 40
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1
www.catvirus.com
Feline Infectious Disease Masterclass
Dr Diane D. Addie
__________________’s workbook
page
SATURDAY
Diagnosis of effusive FIP using an algorithm lecture 2
Dr Soma’s probability of wet FIP table 3
Case history: Mirabelle 4
Case history: Jess 8
Workshop 1: effusive FIP diagnosis
11
Case history: did this cat get FIP when you spayed her? 11
Case history: does Lisa have FIP? 12
Case history: does Oliver have FIP? 17
SUNDAY
Non-effusive FIP lecture 18
Workshop 2: non-effusive FIP diagnosis
Case history: does Zena have FIP? 23
Case history: does Buffy have FIP? 24
Case history: does Tommy have FIP? 25
Case history: does Basil have FIP? 29
Recommended laboratories and contacts 30
References 30
APPENDICES AND SPARE WORKSHEETS
Catvirus.com FIP diagnosis worksheet template 33
FIP diagnostic flowchart Step 1 for the cat’s guardian to complete 34
Treatment protocol for effusive FIP 35
Treatment protocol for non-effusive FIP 36
Effusive FIP diagnosis algorithm 37
Non-effusive FIP diagnosis flowchart 38
FCoV GIT diagnosis flowchart 39
Spare flowcharts for use in the workshop 40
Diane D. Addie GIEFEL October 2016
www.catvirus.com
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Diagnosing effusive FIP using an algorithm
“A wrong diagnosis can be far more devastating than no diagnosis.” Dr Mike
Willard
“More cats have died of FIP tests than have died of the disease.” Dr Niels Pedersen
In most cases, FIP is fatal. An erroneous diagnosis of FIP can be tragically fatal: if an
inappropriate therapy is given or if guardians opt to euthanase their pet to avoid
suffering, thus the life of the pet is unnecessarily wasted.
In the first edition of my book for cat guardians, Feline Infectious Peritonitis and
Coronavirus, I made the statement that 80% of cats diagnosed with FIP turn out to
have some other – often treatable – disease. My statement was based on having had a
summer student telephone veterinary surgeons who had submitted samples to our
diagnostic laboratory at the University of Glasgow Veterinary School, backed up by
my personal experience of getting to the correct diagnosis in cases submitted to me
for second opinion. Various recent publications have enabled me to refine my 80%
figure considerably. While 80% remains true for non-effusive FIP, recent research
has shown that the probability that a cat really has effusive (wet) FIP varies with the
cat’s age and breed.
The probability that an effusion IS caused by FIP varies with the age and breed
of the cat: between 5% and 89% of cats with effusions suffer from some other
condition
Effusive FIP is much more easy to diagnose than non-effusive FIP. The legendary
Italian veterinary pathologist, Dr Saverio Paltrinieri, published a paper in which 79 of
110 cats with effusions (72%) were diagnosed as having wet FIP, in the other 31 cats,
the effusions were due to diseases other than FIP. Thus a correct diagnosis was more
likely to be obtained in effusive FIP compared with only around 20% (or less Jeffery et al,
2012) correct diagnoses of non-effusive FIP.
Recent research indicates that a positive FCoV RT-PCR test on an effusion is 100%
diagnostic of FIP. Doenges et al; Felten et al; Longstaff et al.
In 2013, Dr Soma and his colleagues
published results of FCoV RT-PCR tests on an enormous number of effusions sent to
his laboratory in Japan: these results showed that the percentage of effusions positive
by FCoV RT-PCR varied with the cat’s breed and age, see table 1. which I have
adapted from the graph published in Dr Soma’s paper. Up to the age of 4-5 years,
purebred cats were more likely to be positive than domestic cats, and after 6 years of
age the converse became true (possibly because the domestic cats have experienced
exposure to FCoV in a rescue or boarding cattery). The percentage of effusions
positive for FCoV decreased with the age of the cat: from 95% of 139 effusions from
pedigree cats up to one year old, to only 11 % of effusions from pedigree cats of 10
years of age or older.
Diane D. Addie GIEFEL October 2016
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Table 1. Likelihood of effusive FIP according to age and breed Soma et al
Age (yrs) Pedigree cat Domestic cat
<1 95 79
1 70 42
2-3 53 41
4-5 60 33
6-7 20 21
8-9 23 34
≥ 10 11 14
In this table, the likelihood of a cat with an effusion having FIP is given: for
example, a pedigree cat under a year of age is 95% likely to have FIP, whereas a
domestic cat aged 6-7 years of age is only 21% likely to have FIP. From this
table, you can work out the probability of effusive FIP according to a cat’s age
and breed.
Obviously this table is NOT a substitute for sending a sample of effusion to a
reputable veterinary laboratory for a FCoV RT-PCR test.
If you want to, you can read a full blog I wrote on this subject at:
The presence of antibodies indicates only that the cat has been infected with
FCoV, the cause of FIP, not that the cat has FIP: bear in mind that the cat may be
sick with some non-FIP disease and co-incidentally infected with FCoV. Most cats
with FIP have extremely high antibody titres, but any FCoV antibody titre can occur
in cases of effusive FIP. Antibody titres of 0 are unusual in FIP cases and are usually
considered as indicating that the cat does not have FIP. (However, see below.)
An independent comparison of FCoV antibody tests showed that the best test was the
FCoV Immunocomb (Biogal, Israel). Addie et al, 2015
This test is ideal for the larger
veterinary hospital with its own small laboratory, the test kit comes complete and is
stored in the fridge. However, it does take around 45 minutes to perform. The best
rapid immunomigration (RIM) test was the F-Corona from Virbac. However,
sensitivity was a problem with some other commercially-available tests. False
positive results were rarer than false negative results and only came from some
laboratories using TGEV for immunofluorescence.
Don’t use effusions on RIM tests: doing so can give in false negative results
The Speed F-Corona (Virbac, France) and FASTest FIP (MegaCor, Austria) were the
best rapid immunomigration (RIM) tests for FCoV antibodies. Addie et al, 2015
Sensitivity
of RIM tests was sometimes adversely affected by using them with an effusion: we
showed that increasing amounts of virus in an effusion caused a decrease in antibody
signal, presumably by binding of the antibody in the sample with virus in the sample,
making it unavailable to bind with viral antigens in the test, causing a false negative
result. Meli et al
In such cats, where FIP is strongly suspected on clinical grounds,
despite negative FCoV serology, FIP can be confirmed by FCoV RNA detection (RT-
PCR), performed on a sample of the effusion (see step 4).
Step 4: Effusive (“wet”) FIP – sending the effusion to a veterinary
laboratory
Key message: a positive FCoV antibody test does NOT mean that a cat has FIP:
only that he or she has been exposed to FCoV infection
Key message: a positive FCoV RT-PCR test on an effusion is diagnostic of FIP.
However, a negative result does not rule out FIP: it depends on the sensitivity of the
RT-PCR test
Diane D. Addie GIEFEL October 2016
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Since the advent of reverse-transcriptase polymerase chain reaction (RT-PCR) testing
becoming commercially available in many countries, diagnosis of effusive FIP has
become relatively straightforward: have FCoV RT-PCR tested on the effusion!
Unless the test has poor specificity (e.g. the primers for RT-PCR for a FCoV
messenger RNA RT-PCR Simons et al, 2005
also recognised some human DNA) then a
positive result will be confirmation that the cat has effusive FIP, Doenges et al; Felten et al;
Longstaff et al especially if a quantitative RT-PCR was used and a large amount of virus
detected. However, at time of writing, no paper has been published comparing the
sensitivities of the various different commercially available FCoV RT-PCR tests: thus
a negative test may not be able to rule out FIP. Since FCoV is an RNA virus, it is
highly subject to mutations, which mean that designing primers and probes for RT-
PCRs can be challenging: a conserved region of the genome should be chosen.
Veterinary surgeons should find out which RT-PCR their reference laboratory uses
and try to choose a test which has been published in peer-reviewed literature: a list of
laboratory tests this author trusts is given towards the end of these notes.
Sending an effusion to a veterinary laboratory for FCoV RT-PCR
Only a small amount of effusion is required for RT-PCR testing: 1ml in a plain tube
will certainly give enough for a laboratory to come up with a result. Although FCoV
is an RNA virus, and RNA is quite fragile, in fact when it is within a biological
sample, such as an effusion, or faeces, it is remarkably robust, and can be sent in
ordinary mail, without ice, without loss of a signal, for up to 3 weeks.
If using the University of Glasgow Veterinary Diagnostic Services laboratory it is
worth taking advantage of the amazing cytologists who also work there, and who can
often give you a diagnosis for samples which are negative, so include an air dried
smear of the effusion, and some effusion in an EDTA tube.
Diane D. Addie GIEFEL October 2016
www.catvirus.com
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EFFUSIVE FIP WORKSHOP
CASE HISTORY: DID THIS CAT CATCH FIP IN YOUR SURGERY?
You spay a young pedigree cat and when she comes back for her stitches out, you
notice that her abdomen is enlarged and feels fluidic.
1. Has she caught FIP while in your veterinary surgery????
2. List your explanations of what may have happened.
3. What steps are you now going to take to establish a diagnosis?
4. What steps will you take to ensure that this does not occur again?
Diane D. Addie GIEFEL October 2016
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CASE HISTORY: DOES LISA HAVE FIP?
Steps 1 & 2: history and clinical signs
An email from Lisa’s guardian said this:
“Our nearly 5 year old Lisa (Russian Blue), the youngest
of our 4 cats, got diagnosed with wet FIP 2 weeks ago, on
Monday, 18 July 2016. She was in a very bad shape,
breathing heavily, but she improved the next day after
about 130ml fluid has been drained out from her chest.
She was put on antibiotics (Novoclad) straight away and
kept improving. A week later we started giving her
Prednisolone (1 a day) in addition to the antibiotics.
Yesterday, exactly two weeks later our vet was surprised
to see her going that well as usually the wet FIP cases die
in within a few days after diagnosis.
Lisa has been eating well all the time even when she was
very sick but now she has been playful again and alert and
appears to be her
normal self again. She has been breathing a bit faster than our other cats but this
could be due to the remaining fluid that her body is fighting to get rid of, the fluid was
not drained out completely. She sleeps on her side and seems pretty relaxed.
Our vet said the type of fluid pretty much indicates FIP. The Rivalta Test was
positive. The PCR test was not done as we thought we might not have 2 weeks left to
wait for the test result. Also, being told the test would give us only a 75% probability
we decided to start with the antibiotics instead and see how we go.”
Question 1. Has this cat had the opportunity to become infected with FCoV?
Answer …………………………………………………………………………………
Question 2: What is there in the HISTORY part of Rosana’s email which would rule
out, or rule in, a diagnosis of FIP?
Answer ………………………………………………………………………………..
…………………………………………………………………………………………
…………………………………………………………………………………………
Question 3: What is there in the CLINICAL SIGNS part of Rosana’s email which
would rule out, or rule in, a diagnosis of FIP?
Answer
…………………………………………………………………………………………..
………………………………………………………………………………………….
………………………………………………………………………………………….
Diane D. Addie GIEFEL October 2016
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Step 3: In-house effusion results - we don’t have any, but the following results
were obtained from VetPath Laboratory
Step 4: External Laboratory Results
Vet Path Lab Services: CYTOLOGY: PLEURAL FLUID The sample consisted of 2mL of pale yellow, slightly cloudy, slightly viscous fluid. Cell count: 7.52 x 10^9/L Protein: 48 g/L SG: 1.032 Rivalta: positive Cytological preparations show a moderately cellular and fairly well preserved preparation on a stippled, eosinophilic, proteinaceous background which contains small numbers of erythrocytes. Nucleated cells consist predominantly of non-degenerate neutrophils. There are lesser numbers of large macrophages and occasional small lymphocytes. The macrophages occasionally demonstrate recent erythrophagia, and there is also phagocytosis of degenerate cellular/nuclear material. No overtly malignant cells or infectious agents are identified. Cytological preparations from the right thoracic fluid are very similar, although a slightly greater percentage of macrophages is present. Protein concentrations, and nucleated cell count are similar to those encountered for the left thorax. Neutrophils: 88 % Lymphocytes: 2 % Macrophages: 10 % Eosinophils: 0 % Gram Stain: no bacteria seen INTERPRETATION: Exudate COMMENT: This fluid is classified as an exudate, and the positive Rivalta test indicates an underlying inflammatory aetiology. The cell count is lower than would typically be associated with pyothorax, however, an atypical bacterial infection such as Mycobacteria may be associated with a relatively low cell count. Other considerations include FIP, and an underlying necrotic/ischaemic process as may be encountered with neoplastic disease, or a diaphragmatic hernia.
FCoV antibody titre: 1:2560
Toxo IgG 1:256
Toxoplasma IgM: < 1:16
FIV: Negative University of Sydney report on examination of the pleural fluid: Direct Immunofluorescence Report
Several cell preparations (cytospins) were made from the fluid submitted. A protein level of 55 g/L was measured on the fluid. Using a fluorescin labelled antibody against Feline Coronavirus (types I and II), immunofluorescence was performed to identify the presence of the virus within macrophages seen in the fluid. This was NEGATIVE for feline coronavirus infected macrophages in the fluid.
Diane D. Addie GIEFEL October 2016
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19 July 2016 haematology and biochemisty from VetPath Laboratory
Diane D. Addie GIEFEL October 2016
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12 August 2016
Diane D. Addie GIEFEL October 2016
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Diane D. Addie GIEFEL October 2016
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CASE HISTORY:
DOES OLIVER HAVE FIP?
Cat’s name ............... Oliver ............................................................ .........................
precipitates, haemorrhage into anterior or posterior chambers
mesenteric lymph node enlargement
pyogranulomata on the kidneys, leading to renomegaly
a colonic form of non-effusive FIP is recognised presenting with large
intestinal diarrhoea or constipation
Step 3: Non-effusive (“dry”) FIP – haematology and blood biochemistry
Haematology
In non-effusive FIP there is often lymphopenia; a mild non-regenerative anaemia with
a haematocrit of 30% or less (becoming severe as the FIP progresses); sometimes a
neutrophilia with a shift to the left. Unfortunately these haematological changes are
common to a vast array of chronic diseases in the cat, not just FIP. Haematology
examination is especially useful in differentiating FIP from feline infectious anaemia
infection where the anaemia is regenerative and there may be haemotropic
Mycoplasma spp organisms on the erythrocytes visible in a blood smear. Other
indicators of infectious anaemia as the diagnosis, rather than FIP, are an enlarged
spleen, extremely high temperatures (104-5oF, 40oC) cycling at 7-10 days, and a
response to doxycycline treatment.
Hypergammaglobulinaemia resulting in low albumin:globulin ratio (A:G) ratio
In FIP the globulin concentration in serum or plasma is raised to over 45g/l.
Consequently the A:G is usually lowered. An A:G of < 0.4 indicates FIP is quite
likely, provided that globulins are raised. However, remember than a low albumin
(e.g. in liver disease) can also artificially lower the A:G, so normal globulin likely
rules out FIP even if the A:G is low.
An A:G of >0.8 rules out FIP. A:G of between 0.4-0.8 is inconclusive, so consider
other parameters.
Bilirubin levels are often raised, although other liver parameters may be normal.
Rising bilirubin levels are a poor prognostic sign.Tsai et al
Key message: a negative FCoV antibody test is useful in ruling out FIP.
(Provided the test is sensitive enough.)
Diane D. Addie GIEFEL October 2016
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A negative FCoV antibody test, provided the test is a good quality test with good
sensitivity, rules out non-effusive FIP (see FCoV antibody test section above). FCoV
antibody titres in dry FIP are usually extremely high.
Note: many healthy cats and cats with diseases other than FIP have FCoV
antibodies. The presence of FCoV antibodies alone is NOT diagnostic of FIP, if the
other parameters of the profile do not indicate a diagnosis of FIP.
I am quite appalled at how often a diagnosis of FIP is made on the basis of a high
FCoV antibody titre alone with absolutely NO other supporting evidence for such a
diagnosis.
On CSF analysis, the presence of FCoV antibodies is possibly more useful for
diagnosing neurological FIP than is the detection of viral RNA by RT-PCR since the
latter is sometimes positive in non-FIP cases.
Step 4: specialised laboratory tests
Alpha one acid glycoprotein
Alpha one acid glycoprotein (AGP) is an acute phase protein which has been shown
to be very useful in distinguishing FIP from other clinically similar conditions. Cats
with non-effusive FIP tend to have lower AGP levels than cats with effusive FIP, but
it is still at least twice normal (normal is up to 500 μg/ml). However, AGP rises in any
infectious or inflammatory condition, and also after surgery.
In non-infectious liver disease and neoplasia, which are the most common conditions
mistaken for non-effusive FIP, AGP is usually normal.
Pathology and histopathology on the deceased cat
Histopathology is generally regarded as the gold standard of FIP diagnosis:
histopathologists look for a perivascular pyogranuloma. In non-effusive FIP there
tends to be fewer (but often larger) lesions than in effusive FIP and often a full
exploratory laparotomy or post mortem is required to find the lesions. Grossly, FIP
lesions can be indistinguishable from tumours, necessitating histopathology to
differentiate. Some cats only have lesions in the brain, spinal cord or eye which
necessitates special instruments to access.
Sending an eye for histopathology: for light microscopy: Davidson's solution or
Bouin's solution are the routine fixatives used for the eye. Either of these provides
adequate preservation of tissues and should be used for all globes. Formalin should
not be used on globes if this can be avoided because it does not provide adequate
Key message: positive FCoV serology is NOT DIAGNOSTIC OF FIP
Diane D. Addie GIEFEL October 2016
www.catvirus.com
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preservation of the retina in particular. It should be limited to adnexal tissue. www.vetmed.ucdavis.edu/courses/vet_eyes/eye_path/epath_overview_index.html
Sending the entire body to the laboratory is more likely to result in diagnosis
than sending organ samples
Cave et al 2002
reported that sending an entire deceased kitten to the pathology
laboratory at Glasgow Veterinary School was more likely to result in a diagnosis than
simply sending samples of organs.
Biopsy /Trucut/ Punch biopsy / Fine needle aspirate
FIP can be successfully diagnosed by observing lesions typical of FIP in smear
preparations of FNAs or Tru-cut biopsies of the liver or kidney, although there was a
problem that many of the samples were inadequate (the cytology ruined) which meant
sensitivity was relatively low. Giordano et al, 2005
However, I have an ethical problem with
putting a cat who may be doing through such an invasive procedure.
FCoV can be detected in a fine needle aspirate of the mesenteric lymph node Kwok
et al, manuscript in preparation It is wise, if you are taking fine needle aspirates or biopsies for
FIP diagnosis to divide your samples into two and put one into 0.25ml saline, not
formaldehyde, so that FCoV RT-qPCR can be performed if histopathology doesn’t
give you an answer. Store the FNA in the fridge or freezer if you do not want to do
FCoV RT-PCR on it immediately: FCoV RNA will remain detectable for many weeks
there. Addie personal observation
Detection of virus by immunohistochemistry: can get false positives
Immunohistochemistry (IHC) is used to demonstrate the presence of virus in the
lesions of FIP, it has been considered the absolute gold standard in FIP diagnosis,
although I believe it will be surpassed by RT-PCR tests. It can be a useful
confirmatory test in cases in which the histologic findings are not typical of FIP.
However, it is essential that the correct controls are in place (i.e., that a non-FCoV
antibody is used as a control on every organ section being examined, since feline
tissue is sticky and will often non-specifically bind irrelevant antibody, for example
the conjugated antibody being used to detect the antibody detecting the coronavirus).
Lack of these controls will result in false positive diagnoses of FIP and a large chain
of veterinary laboratories has been known to give false positive diagnoses of FIP
using this technique.
RT-PCR for detection of FCoV RNA
Reverse transcriptase polymerase chain reaction (RT-PCR) detects the RNA of the
FCoV: i.e. is a test which detects presence of actual virus. Quantitative RT-PCR
(RT-qPCR) allows the amount of virus in the sample may be measured. Confusion
can arise because quantitative RT-PCR is sometimes referred to as “real time” which
may also be shortened to RT. In the early days it was referred to as Taqman PCR after
the first quantitative PCR machine. RT-PCR of faecal samples is useful in control of
FCoV infection in households of healthy cats and RT-PCR is useful in FIP diagnosis
on organs of cats in biopsy or post mortem specimens.
Diane D. Addie GIEFEL October 2016
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In non-effusive FIP, detection of large amounts of virus in a fine needle aspirate of a
mesenteric lymph node is highly indicative of FIP Kwok et al, manuscript in preparation.
Blood samples are usually negative, so testing them is not useful. Detection of FCoV
RNA in the faeces is not diagnostic of FIP, since around 33% of healthy seropositive
cats, or animals with non-FIP illness, are also positive.
CSF
Detecting FCoV in the CSF of cats is not diagnostic of FIP: healthy cats and cats with
non-FIP conditions are occasionally positive (detecting FCoV antibody in the CSF
may be more useful).
Key message: DO NOT get FCoV RT-PCR tests on blood: they are a waste of
time and resources
Diane D. Addie GIEFEL October 2016
www.catvirus.com
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CASE HISTORY: DOES ZENA HAVE FIP?
Cat’s name ...........Zena................................................................ ..................................