Feline Infectious Disease Masterclass Dr Diane D. Addie. s workbook

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1 Feline Infectious Disease Masterclass Dr Diane D. Addie s workbook 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 page 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 1

2 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 Jeffery et al, likely to be obtained in effusive FIP compared with only around 20% (or less 2012 ) correct diagnoses of non-effusive FIP. Recent research indicates that a positive FCoV RT-PCR test on an effusion is 100% Doenges et al; Felten et al; Longstaff et al. diagnostic of FIP. 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. 2

3 Table 1. Likelihood of effusive FIP according to age and breed Age (yrs) Pedigree cat Domestic cat < Soma et al 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: If you do read it please give it an upvote! Most cats infected with FCoV do not develop FIP 3

4 The cause of FIP is infection with feline coronavirus (FCoV). However, most FCoV infections do not have any serious consequences: the majority of infected cats have subclinical infection, or a bout of diarrhoea. A small percentage of cats mounts a deleterious immune response to FCoV: clinical signs form a spectrum from very acute severe infection with destruction of many blood vessels and leakage of plasma into body cavities this is known as effusive, or wet, FIP and death within days to weeks, to chronic or non-effusive FIP, which can last for months. (See my YouTube animation ). In dry or non-effusive FIP the progress is slower, the number of blood vessels damaged is fewer, and a chronic immune response is a pyogranuloma formation as the body attempts to wall off the infection. Non-effusive FIP is much more difficult to diagnose than effusive FIP. The effusive FIP diagnosis algorithm, the step one questionnaire and worksheet templates are in the Appendices. The FCoV awareness / pedigree kitten poster is in a separate document. All are available for download from Key message: Feline infectious peritonitis (FIP) is a disease predominantly affecting young pedigree (purebred) kittens and cats. However, any age of cat can be affected. CASE HISTORY 1: MIRABELLE Cat s name... Mirabelle Breed... Age...Sex...F Other cats?... Indoor/Outdoor How could this cat have gotten FCoV infection? How long ago might the cat have become infected? (if it was more than 18 months, this is unlikely to be FIP) Recent stress? Clinical signs

5 Step 1. Question 1. Where/how could your cat have caught FCoV? FIP can only develop in a cat who has been infected with feline coronavirus (FCoV) it cannot occur out of the blue. Go through the following questions to try to establish whether or not your cat has had the chance to become infected. Score through or circle the Yes or No My cat is pedigree (purebred) I have more than 6 cats and they use litter trays My cat came from a rescue shelter My cat has been in a boarding cattery We recently obtained a new cat or kitten My cat has been to a cat show in the last year My cat has visited a stud cat in the last year A queen visited my stud cat in the last year If the answer is Yes to any of the questions above, then your cat has possibly had the opportunity to become infected with FCoV. If you answered No to all of the questions above, then FIP seems unlikely, but is not completely ruled out. Step 1. Question 2. Has the cat experienced a stress in the last 18 months? We only got the cat within the last year My cat has or had another illness recently We recently obtained a new cat or kitten We recently got a dog or puppy We recently had a baby or adopted a child We have 6 or more cats We have moved house in the last year We put the cat into a boarding cattery Somebody the cat loves has been away, or ill, or died The cat recently gave birth We took our cat to a cat show The cat was hospitalised at the veterinary surgery The cat has been on some other kind of journey The cat has been exposed to some other stress not listed here If the answer is yes to any of the questions above, then your cat has likely experienced stress. If you answered No to all of the questions, try to think if your cat has experienced a stress which I have not listed. If you answered no to all the questions in step 1, FIP seems very unlikely: take your answers and the algorithm to your veterinary surgeon and discuss the situation with him or her. Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because

6 Treatment / next diagnostic action... Step 2: Effusive ( wet ) FIP - clinical signs Key message for step 2, clinical signs: CATS WITH FIP HAVE SOME ABNORMAL CLINICAL SIGNS Cats with FIP are not simply fat or pregnant! In effusive FIP, there is an effusion! Effusive FIP is the more acute condition than dry FIP: usually occurring within days to weeks of FCoV infection and/or a stressful event in the cat s life. FIP is an immune-mediated vasculitis: in effusive FIP, many blood vessels are affected, allowing fluid to leak out into the abdomen, thorax or pericardium. Thus the cat presents with ascites or pleural and occasionally pericardial effusion. The ascitic cat may appear to have put on weight, although ribs are usually more palpable. The cat may still be bright and eating, though some are dull and anorexic. The temperature of cats with FIP rarely exceeds 103 o F (39 o C). A cat with a pleural effusion will present with dyspnoea. Cats with a pericardial effusion will have muffled heart sounds. The single most useful thing you can do next is to analyse the effusion. Step 3: Effusive ( wet ) FIP - analysis of effusion Drawing off the effusion is useful for 3 reasons: it relieves some of the clinical signs for the cat, removing not just physical fluid, but also virus and sources of inflammatory cytokines it enables a far more accurate diagnosis than a blood sample it allows you to place a needle for administering interferon omega or other treatment directly to the site of the lesions Paracentesis can often be performed in the conscious cat using only clipnosis Pozza et al (applying clothes pegs or paper clips to the scruff of the neck). Look at the effusion and smell it if it stinks, you are dealing with a bacterial infection, NOT FIP FIP effusions vary in appearance: most are clear and straw-coloured and froth when shaken (because of the high protein content). Some may be blood-tinged, or even lipaemic, but cloudy, turbid, stinking purulent effusions point to a bacterial infection, not FIP. A negative Rivalta test is 93% NOT likely to be effusive FIP The Rivalta test is one of the simplest and most economic ways to rule out FIP (the test has a high negative predictive value (NPV) of 93.4% for FIP), costing pennies to perform and taking only a couple of minutes. To perform a Rivalta test, one drop of 8% acetic acid (ordinary clear/white vinegar) is added to 5 10 mls of still water (which must be at room temperature) in a clear test tube and mixed thoroughly. A 6

7 drop of effusion is carefully layered on top. If the effusion dissipates like a wisp of smoke in air the Rivalta test is negative and the cat is 93% not likely to have FIP. Fischer et al, 2012 If, however, the effusion hangs from the surface in a globule, then slowly floats down like a jellyfish, the Rivalta test is positive. A positive Rivalta test means that the cat is 58.4% likely to have FIP (i.e. only about 6 of 10 cats with a positive Rivalta test do have FIP, the other 4 cats have some other condition). Positive Rivalta s test results may also be obtained in cats with bacterial peritonitis or lymphoma. However it is usually easy to differentiate these effusions by macroscopic examination, cytology and/or bacterial culture. You can watch a video of positive and negative Rivalta tests on my YouTube channel ; however please be aware that the video was made before a more recent publication by Fischer et al, 2012 and that in the film I give percentages from a previous publication by Hartmann et al, 2003 of PPV of 86% and NPV of 97% which were over optimistic. One problem with the Rivalta test is that it is quite subjective: a blinded study showed that two independent investigators Fischer et al, 2012 gave quite different interpretations. In addition, around 10% of Rivalta tests cannot be conclusively designated as either positive or negative. Fischer et al, 2012 I found that I needed to practise with quite a few effusions, and that the water that I used considerably altered the results, curiously my own tap water (which is from a mountain spring) gave better results than distilled water. Use water at room temperature or even slightly warmed rather than from a fridge (cold water causes false negative reactions). The test can work with old effusion samples which have been stored in a fridge for some days but can give false negative results if the effusion has been frozen. Total protein in the effusion and albumin:globulin ratio (A:G) The total protein concentration in the effusion of a cat with FIP is usually greater than 35 g/l and this usually consists of more globulin than albumin, pushing down the albumin to globulin (A:G) ratio. The A:G of an effusion is one of the most useful tests to perform in practice for a quick indicator of whether or not a cat may have FIP and can be easily performed on an in-house biochemistry analyser machine. To calculate the A:G ratio, divide the albumin by the globulin values. An A:G of < 0.4 indicates FIP is quite likely; an A:G of >0.8 rules out FIP; A:G of between is inconclusive so consider other parameters. At this stage, you will be able to rule out effusions due to cardiomyopathy which is a major differential in the young cat: such effusions are transudates, and usually have only a few grams of protein per litre, although when an effusion has been present for a long time, it begins to irritate the serosae and can become an exudate. Cytology In effusive FIP, there are generally fewer than 3 x 10 9 nucleated cells per litre in the effusion (i.e. the effusion is a modified transudate, although around 10% of FIP effusions are more cellular). In FIP, the cells are predominantly neutrophils and macrophages. In bacterial peritonitis and pleurisy, the white blood cell count in the effusion is much higher and the cytologist will usually see bacteria (if bacteria are intracellular, this indicates that they were not simply contamination of the sample). 7

8 Cytology of pleural effusions is useful for differentiation of thymic lymphosarcomas, since the predominant cell is the lymphocyte and they often appear malignant: in FIP effusions lymphocytes are uncommon. CASE HISTORY: JESS Cat s name... Jess Breed...DSH... Age...12 years...sex...female neutered... Other cats?...no... Indoor/Outdoor How could this cat have gotten FCoV infection?... Clinical signs... How long ago might the cat have become infected? (if it was more than 18 months, this is unlikely to be FIP) Recent stress? Test Blood Effusion Unknown/ Comment on significance Other Albumin 26 Globulin 22 Alb:glob Bilirubin AGP FCoV antibody titre FCoV RT- PCR Hct Lymphocytes 0.43 x 10 9 /l 72% of the wbc in the effusion were lymphocytes Cytology NA Other NA = not applicable Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because

9 Treatment / next diagnostic action... FCoV antibody test 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 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 Meli et al result. 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 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 9

10 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 Doenges et al; Felten et al; positive result will be confirmation that the cat has effusive FIP, 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. 10

11 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? 11

12 Steps 1 & 2: history and clinical signs CASE HISTORY: DOES LISA HAVE FIP? An 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 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 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 which would rule out, or rule in, a diagnosis of FIP? Answer

13 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: 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. 13

14 19 July 2016 haematology and biochemisty from VetPath Laboratory 14

15 12 August

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17 CASE HISTORY: DOES OLIVER HAVE FIP? Cat s name... Oliver Breed... Age...9m...Sex...Male neutered... Other cats?... Indoor/Outdoor...Single cat household... How could this cat have gotten FCoV infection? How long ago might the cat have become infected? (if it was more than 18 months, this is unlikely to be FIP) Recent stress? Clinical signs... Presentation: dyspnoea... Respiratory rate: 72 bpm (tachypnoeic)... Auscultation: respiratory sounds reduced... Mucous membranes: pale... Test Blood Effusion Unknown/ Other Albumin 22 Globulin 75 Alb:glob Bilirubin AGP 1900 FCoV >1280 antibody titre FCoV RT- Not Positive PCR advised Hct Lymphocytes Cytology NA Other NA = not applicable Comment on significance 17

18 Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because... Treatment / next diagnostic action NON-EFFUSIVE FIP Key message: 80% of cats diagnosed with non-effusive FIP turn out to have some other condition Step 1: Non-effusive ( dry ) FIP history Step 1 is pretty much the same as for effusive FIP, except that the incubation time from becoming infected with FCoV, and even from the stressor, may be much longer many weeks, even months. Step 2: Non-effusive ( dry ) FIP clinical signs Key message: cats with non-effusive FIP are not clinically well! Cats with non-effusive FIP do NOT have a normal temperature, normal appetite, they are not bright and responsive. Too many healthy cats with positive FCoV antibody or RT- PCR tests have been erroneously diagnosed as having dry FIP and euthanased. Non-effusive FIP is the more chronic of the two forms of FIP, incubating months to even years after the initial FCoV infection and the triggering stress. The dry FIP cat loses weight gradually, is chronically or intermittently pyrexic (up to 103 o F / 39 o C), and becomes dull and anorexic. Most cats with dry FIP also have palpably enlarged mesenteric lymph nodes and intraocular lesions, although the latter may be quite subtle and require a thorough examination to detect. If you understand that FIP is an immune-mediated vasculitis it becomes easier to understand how it is able to manifest with so many varied clinical signs. Any blood vessel to any organ can be affected and the clinical signs will result from the impaired 18

19 blood supply and the developing granulomata in that organ. In non-effusive FIP, fewer blood vessels are affected and the immune response is more chronic, leading to larger pyogranulomata, which may even be mistaken for tumours. Clinical signs depend on which organs are involved, examples include: liver infiltration leading to jaundice meninges/hydrocephalus leading to neurological signs (ataxia, nystagmus, seizures, loss of reflexes); pyogranulomata around a nerve/ spinal column leading to neurological signs eyes: uveitis, aqueous flare, vitreous flare, retinal vessel cuffing, corneal 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 is inconclusive, so consider other parameters. Bilirubin levels are often raised, although other liver parameters may be normal. Tsai et al Rising bilirubin levels are a poor prognostic sign. Key message: a negative FCoV antibody test is useful in ruling out FIP. (Provided the test is sensitive enough.) 19

20 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. Key message: positive FCoV serology is NOT DIAGNOSTIC OF FIP 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 20

21 preservation of the retina in particular. It should be limited to adnexal tissue. 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 Addie personal observation there. 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. 21

22 In non-effusive FIP, detection of large amounts of virus in a fine needle aspirate of a Kwok et al, manuscript in preparation. mesenteric lymph node is highly indicative of FIP Key message: DO NOT get FCoV RT-PCR tests on blood: they are a waste of time and resources 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). 22

23 CASE HISTORY: DOES ZENA HAVE FIP? Cat s name...zena Breed...Maine Coon... Age...9m...Sex Other cats? Buffy...Indoor/Outdoor Recent stress? Clinical signs...keratic precipitates Test Blood Effusion Normal range Albumin 23 g/l NA g/l Globulin 91g/l NA g/l Alb:glob 0.25 NA Bilirubin AGP 1400 microg/ml >1280 up to 500 µg/ml FCoV antibody titre FCoV RT- PCR Hct Lymphocytes Cytology NA Other NA = not applicable Comment on significance Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because Treatment/next diagnostic action What is Zena s prognosis?

24 CASE HISTORY: DOES BUFFY HAVE FIP? Cat s name...buffy Breed...Maine Coon... Age...1 yr...sex Other cats?...zena...indoor/outdoor Recent stress? Clinical signs...in contact with Zena. Eyes OK Test Blood Effusion Normal range Albumin 38 g/l NA g/l Globulin 32 g/l NA g/l Alb:glob NA Bilirubin AGP 280µg/ml up to 500 FCoV >1280 antibody titre FCoV RT- PCR Hct Lymphocytes Cytology NA Other NA = not applicable Comment on significance Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because Treatment/next diagnostic action What is Buffy s prognosis? What follow up tests might you do?

25 CASE HISTORY: DOES TOMMY HAVE FIP? For each aspect of the FIP diagnosis algorithm, please put: a tick ( ) for any result consistent with FIP a cross (X) for any result against a diagnosis of FIP and if you are not sure, or it s ambiguous, a question mark (?) At the end of working through the algorithm, you should have a preponderance of either ticks or crosses. Step 1: has Tommy had an opportunity to become infected with FCoV? Tommy is a MN DSH of unknown age who came from a 28 cat rescue fosterer in 2006 along with one other cat. He is now in a 3 cat household in a rural area with indoor/outdoor access. The third cat (DSH) was obtained in 2007 from a farm. The cats have not been to a boarding cattery. He was presented in 2009 with the clinical signs seen in the photograph below. Is there anything else you noticed about Tommy s history which could point the way towards his diagnosis? Step 2: Tommy s clinical signs 25

26 In the photograph above you can see Tommy s main presenting signs, and below are his clinical notes for any signs you can t readily obtain from the photo: T Appetite variable Mesenteric lymph nodes not enlarged Mucous membranes did not appear icteric 1. Are you thinking effusive or non-effusive FIP? 2. Are his eyes normal? Describe what you see Step 3: In-house blood results These are photos of the in-house blood results for you to extract the relevant information from: 26

27 Cat s name... Tommy Breed... Age...old...Sex...MN.... Other cats?... Indoor/Outdoor... How could this cat have gotten FCoV infection? Clinical signs... How long ago might the cat have become infected?... (if it was more than 18 months, this is unlikely to be FIP) Recent stress?

28 Test Blood Effusion Unknown/ Other Albumin Globulin Alb:glob Bilirubin AGP FCoV antibody titre FCoV RT- PCR Hct Lymphocytes Cytology NA Other NA = not applicable Comment on significance Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because Treatment / next diagnostic action

29 CASE HISTORY: DOES BASIL HAVE FIP? Cat s name... Basil Breed... Age...Sex...MN... Other cats?... Indoor/Outdoor... How could this cat have gotten FCoV infection?... Basil s history was that he was in contact with 4 other cats who have died of FIP Clinical signs... He presented with jaundice.... How long ago might the cat have become infected?... (if it was more than 18 months, this is unlikely to be FIP) Recent stress?... Test Blood 5/1/04 Albumin 24 Globulin 61 Alb:glob Bilirubin AGP 2600 FCoV 640 antibody titre FCoV RT- Not PCR advised Hct 28.2 Lymphocytes 0.29 Cytology NA Other NA = not applicable Effusion No effusion Unknown/ Other Comment on significance Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because... Treatment / next diagnostic action

30 Recommended suppliers of FCoV antibody tests To obtain a FCoV Immunocomb kit contact Len Small: len@biogal.co.il To obtain F-Corona Speed tests contact your Virbac representative. Recommended laboratories for getting a FCoV RT-PCR test France, Spain: Scanelis Laboratory: Portugal: FACULDADE DE MEDICINA VETERINÁRIA, LABORATÓRIO DE VIROLOGIA E IMUNOLOGIA, Avenida da Universidade Técnica, Polo Universitário da Ajuda, Alto da Ajuda UK: Veterinary Diagnostic Services, University of Glasgow Veterinary School (we receive samples from all over the world): References Addie D.D., Jarrett O A study of naturally occurring feline coronavirus infection in kittens. Vet. Rec Addie D.D., Toth S., Murray G.D., Jarrett O The risk of feline infectious peritonitis in cats naturally infected with feline coronavirus. Am. J. Vet. Res Addie D, Belak S, Boucrat-Baralon C, Egberink H, Frymus T, Gruffydd-Jones T, Hartmann K, Hosie MJ, Marsilio F, Lloret A, Lutz H, Pennisi MG, Radford AD, Thiry E, Truyen U, Horzinek MC Feline infectious peritonitis. ABCD guidelines on prevention and management. J Feline Med Surg. 11 (7) Addie DD, McDonald M, Audhuy S, Burr P, Hollins J, Kovacic R, Lutz H, Luxton Z, Mazar S, Meli M Quarantine protects Falkland Islands (Malvinas) Cats from Feline Coronavirus Infection. J Feline Med Surg Addie DD, le Poder S, Burr P, Decaro N, Graham E, Hofmann-Lehmann R, Jarrett O, McDonald M, Meli ML Utility of feline coronavirus antibody tests J Feline Med Surg 17(2): Cave TA, Thompson H, Reid SWJ, Hodgson DR, Addie DD Kitten mortality in the United Kingdom: a retrospective analysis of 274 histopathological examinations ( ). Vet Rec 151: Cohen TM, Blois S, Vince AR Fatal extraintestinal toxoplasmosis in a young male cat with enlarged mesenteric lymph nodes. Can Vet J 57, 5:

31 Doenges SJ, Weber K, Dorsch R, Fux R, Fischer A, Matiasek LA, Matiasek K, Hartmann K 2015 Detection of feline coronavirus in cerebrospinal fluid for diagnosis of feline infectious peritonitis in cats with and without neurological signs. J Feline Med Surg. Doenges SJ, Weber K, Dorsch R, Fux R, Hartmann K Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis. J Feline Med Surg. [Epub ahead of print] Felten S, Weider K, Doenges S, Gruendl S, Matiasek K, Hermanns W, Mueller E, Matiasek L, Fischer A, Weber K, Hirschberger J, Wess G, Hartmann K Detection of feline coronavirus spike gene mutations as a tool to diagnose feline infectious peritonitis. J Feline Med Surg. [Epub ahead of print] Fischer Y, Sauter-Louis C, Hartmann K Diagnostic accuracy of the Rivalta test for feline infectious peritonitis. Vet Clin Pathol. 41(4): Giordano A, Spagnolo V, Colombo A, Paltrinieri S Changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis (FIP) or exposed to feline coronavirus infection. Veterinary Journal Giordano A, Paltrinieri S, Bertazzolo W, Milesi E, Parodi M Sensitivity of Tru-cut and fine-needle aspiration biopsies of liver and kidney for diagnosis of feline infectious peritonitis. Veterinary Clinical Pathology Giori L, Giordano A, Giudice C, Grieco V, Paltrinieri S Performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases. J Small Anim Pract. 52(3): Hartmann K, Binder C, Hirschberger J, Cole D, Reinacher M, Schroo S, Frost J, Egberink H, Lutz H, Hermanns W Comparison of different tests to diagnose feline infectious peritonitis. J Vet Intern Med. 17(6): Hartmann K, Addie D, Belák S, Boucraut-Baralon C, Egberink H, Frymus T, Gruffydd-Jones T, Hosie MJ, Lloret A, Lutz H, Marsilio F, Möstl K, Pennisi MG, Radford AD, Thiry E, Truyen U, Horzinek MC Toxoplasma gondii infection in cats: ABCD guidelines on prevention and management. J Feline Med Surg. 15(7):631-7 Ishida T, Shibanai A, Tanaka S, Uchida K, Mochizuki M. Use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis. J Feline Med Surg. 2004; 6(2): Jeffery U, Deitz K, Hostetter S Positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population. J Feline Med Surg. 14(12): Korman RM, Cerón JJ, Knowles TG, Barker EN, Eckersall PD, Tasker S Acute phase response to Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' infection in FIV-infected and non-fiv-infected cats. Vet J. 193(2):433-8 Legendre AM, Bartges JW. Effect of Polyprenyl Immunostimulant on the survival times of three cats with the dry form of feline infectious peritonitis. J Feline Med Surg 2009;

32 Longstaff L, Porter E, Crossley VJ, Hayhow SE, Helps CR, Tasker S Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis JFMS in press Meli ML, Burr P, Decaro N, Graham E, Jarrett O, Lutz H, McDonald M, Addie DD Samples with high virus loads cause a trend toward lower signal in feline coronavirus antibody tests. J Feline Med Surg Paltrinieri S, Parodi MC, Cammarata G In vivo diagnosis of feline infectious peritonitis by comparison of protein content, cytology, and direct immunofluorescence test on peritoneal and pleural effusions. J Vet Diagn Invest. 11(4): Porter E, Tasker S, Day MJ, Harley R, Kipar A, Siddell SG, Helps CR Amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis. Vet Res. 45:49. Pozza ME, Stella JL, Chappuis-Gagnon AC, Wagner SO, Buffington CA Pinchinduced behavioral inhibition ('clipnosis') in domestic cats. J Feline Med Surg. 10(1):82-7. Riemer F, Kuehner KA, Ritz S, Sauter-Louis C, Hartmann K 2015 Clinical and laboratory features of cats with feline infectious peritonitis - a retrospective study of 231 confirmed cases ( ). J Feline Med Surg. Ritz S, Egberink H, Hartmann K. Effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis. J Vet Intern Med. 2007; 21(6): Rohrer C, Suter PF, Lutz H The diagnosis of feline infectious peritonitis (FIP): a retrospective and prospective study. Kleinterpraxis Simons FA, Vennema H, Rofina JE, Pol JM, Horzinek MC, Rottier PJ, Egberink HF A mrna PCR for the diagnosis of feline infectious peritonitis. J Virol Methods. 124(1-2): Soma T, Wada M, Taharaguchi S, Tajima T Detection of ascitic feline coronavirus RNA from cats with clinically suspected feline infectious peritonitis. J Vet Med Sci. 75(10): Tsai HY, Chueh LL, Lin CN, Su BL Clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in Taiwan. J Feline Med Surg. 13(2):

33 Feline Infectious Peritonitis (FIP) diagnosis worksheet Dr Diane D. Addie Cat s name Breed... Age...Sex Other cats?...indoor/outdoor Recent stress? Clinical signs Test Blood Effusion Unknown/ Other Albumin Globulin Alb:glob Bilirubin AGP FCoV antibody titre FCoV RT- Not PCR advised Hct Lymphocytes Cytology NA Other NA = not applicable Comment on significance Differential diagnosis What can I do to differentiate from FIP? Conclusion: I believe/do not believe this cat has FIP because

34 FIP diagnostic flowchart Step 1 for the cat s guardian to complete Download the FIP diagnosis algorithm from to take to your veterinary surgeon. You can help your vet by filling in this questionnaire first and giving it to him or her. The tables are only step 1 of the FIP diagnosis algorithm and are designed simply to save time in the veterinary surgery and to help you to give your veterinarian history relevant to make a correct diagnosis. Step 1. Question 1. Where/how could your cat have caught FCoV? FIP can only develop in a cat who has been infected with feline coronavirus (FCoV) it cannot occur out of the blue. Go through the following questions to try to establish whether or not your cat has had the chance to become infected. Score through or circle the Yes or No My cat is pedigree (purebred) I have more than 6 cats and they use litter trays My cat came from a rescue shelter My cat has been in a boarding cattery We recently obtained a new cat or kitten My cat has been to a cat show in the last year My cat has visited a stud cat in the last year A queen visited my stud cat in the last year If the answer is Yes to any of the questions above, then your cat has possibly had the opportunity to become infected with FCoV. 1 If you answered No to all of the questions above, then FIP seems unlikely, but is not completely ruled out. Step 1. Question 2. Has the cat experienced a stress in the last 18 months? We only got the cat within the last year My cat has or had another illness recently We recently obtained a new cat or kitten We recently got a dog or puppy We recently had a baby or adopted a child We have 6 or more cats We have moved house in the last year We put the cat into a boarding cattery Somebody the cat loves has been away, or ill, or died The cat recently gave birth We took our cat to a cat show The cat was hospitalised at the veterinary surgery The cat has been on some other kind of journey The cat has been exposed to some other stress not listed here If the answer is yes to any of the questions above, then your cat has likely experienced stress. If you answered No to all of the questions, try to think if your cat has experienced a stress which I have not listed. If you answered no to all the questions in step 1, FIP seems very unlikely: take your answers and the algorithm to your veterinary surgeon and discuss the situation with him or her. 1 If you said Yes to one of the possible sources of FCoV infection that does not necessarily mean that your cat became infected from that source there is often more than one possible source for becoming infected. 34

35 Protocol for treating effusive FIP Glucocorticoids: Dexamethasone Prednisolone sliding dose: (Anti-inflammatory dose.) Dose 1 mg/kg intrathoracic or intraperitoneal injection sid up to 7d, (stop if effusion disappears sooner) AND: 2 mg/kg/day for days, reducing to 1mg/kg/day for days, then 0.5 mg/kg/day for days, then 0.25 mg/kg/day for days, then 0.25 mg/kg/e.o.d. and so on ceasing after complete remission of clinical signs and return to normal of AGP and globulins If, at any point, the cat s condition regresses, go back to the previous dose. Virbagen Omega: (Virbac) 1 million units/kg into the abdominal or thoracic cavity after draining the fluid, e.o.d reducing to once weekly if remission occurs. Subcutaneous injections can be given instead if preferred, but interferons act locally and every effort should be made to get the treatment as close to the site of infection as possible. *** 35

36 Protocol for treating non-effusive FIP Key message: BE ABSOLUTELY CERTAIN THE CAT HAS FIP BEFORE EMBARKING UPON TREATMENT In some ways this is a more important message in non-effusive FIP than in effusive FIP, since to immunosuppress cats with lookalike infectious conditions such as toxoplasmosis or leishmania would be catastrophic. Corticosteroids Prednisolone sliding dose: (Immunosuppressive dose) Dose 2-4 mg/kg/day for days, reducing to 1-2 mg/kg/day for days, then 0.5 mg/kg/day for days, then 0.25 mg/kg/day for days, then 0.25 mg/kg/e.o.d. and so on ceasing after complete remission of clinical signs If, at any point, the cat s condition regresses, go back to the previous dose. For FIP-related uveitis, topical corticosteroids may also be used. Recombinant feline interferon omega (Virbagen Omega, Virbac) 100,000 U per cat orally s.i.d until AGP, globulins, bilirubin, Hct, lymphocyte count and clinical signs return to normal. Diluting Virbagen Omega (IFN Ω): Virbagen Omega comes in vials of 10 million units. It is reconstituted with 1ml of diluent. To get 100,000 Units/ml, use a 1ml syringe and put 0.1 ml IFN Ω into 5 mls of water or saline: teach the guardian to give 0.5 ml of this per day by mouth. (Store it in the fridge (where it will last up to 3 wks). Divide the remaining IFN into 9 x 0.1ml aliquots and freeze until needed: these syringes will last up to 6 months in the freezer. One 10MU vial will treat a dry FIP cat for almost 3 months. Polyprenyl Immunostimulant Polyprenyl Immunostimulant (Sass & Sass, Inc, Oak Ridge, TN 37830, USA) is a mixture of phosphorylated, linear isoprenols which upregulates biosynthesis of Th-1 cytokine mrnas. It was used successfully in three cats with non-effusive FIP, with survival times of 14 months, over 26 and 27 months, but had no beneficial effect on cats with effusive FIP. Legendre & Bartges, 2009 However, Legendre presented at AAHA that 22% of 58 cats with FIP were alive at 6 months, and only 5% at one year. The dose of 3mg/kg orally is given three times a week until cure or death. I await a report of a controlled clinical trial with interest and suspend judgement until there is more evidence. Concurrent corticosteroids are not advised. One needs permission from the VMD to import it into the UK and it can be purchased from the Vetimmune website. For the latest on PPI availability in Europe and further information on FIP treatment visit the FIP treatment page of my catvirus website. 36

37 EFFUSIVE FIP DIAGNOSIS ALGORITHM 37

38 NON-EFFUSIVE FIP DIAGNOSIS ALGORITHM 38

39 FCOV ENTERITIS DIAGNOSIS ALGORITHM 39

40 40

41 41

42 NON-EFFUSIVE FIP DIAGNOSIS ALGORITHM 42

43 NON-EFFUSIVE FIP DIAGNOSIS ALGORITHM 43