FIP Feline infectious peritonitis
Katrin Hartmann, Prof., Dr. med. vet., Dr. habil., DECVIM
Munich, Germany
Introduction
Feline infectious peritonitis (FIP) is a common disease and a frequent reason for referral; approximately 1 of every 200 new feline cases presented to American Veterinary Teaching Hospitals represent cats with FIP.1 FIP is a fatal, immune-mediated disease, triggered by infection with a feline coronavirus (FCoV). FCoV belongs to the family Coronaviridae, a group of enveloped, positive-stranded RNA viruses that are frequently found in cats. Coronavirus-specific antibodies are present in up to 90 % of cats in catteries and in up to 50 % of those in single cat households. However, only about 5 % of FCoV-infected cats develop FIP in a cattery situation. As FIP is not only common but deadly and has no effective long term management, a rapid and reliable diagnosis is critical for prognostic reasons. Difficulties in definitively diagnosing FIP arise from nonspecific clinical signs, lack of pathognomonic hematological and biochemical abnormalities and low sensitivity and specificity of tests routinely used in practice. It was initially hypothesized that FCoV strains causing FIP are different from avirulent enteric FCoV strains. Those latter strains, however, are antigenetically and genetically indistinguishable and represent virulence variants of the same virus rather than separate virus species. It is now known that cats are infected with the primarily avirulent FCoV that replicates in enterocytes. In some instances, however, a mutation occurs in a certain region of the FCoV genome2 leading to the ability of the virus to replicate within macrophages, which is a key pathogenic event in the development of FIP.
Diagnosis
Diagnosing enteral FCoV can be performed by RT-PCR in feces or electron microscopy of fecal samples. Definitively diagnosing FIP can be extremely challenging in many clinical cases. A weighted score system for FIP diagnosis, taking several parameters into account, including background of the cat, history, presence of clinical sings, laboratory changes, and height of antibody titers, has been suggested.3 This, however, only leads to a certain score or percentage of likelihood of FIP and thus, does not help to definitively confirm the diagnosis. There are, however, certain tests available in the meantime (e. g. staining of antigen in macrophages in effusion or tissue), that, at least in case of a positive result, confirm the diagnosis of FIP 100 %.4
Laboratory Changes
Blood cell counts are often changed in cats with FIP. However, changes are not pathognomonic. Although it is often stated that lymphopenia and neutrophilia are typical for FIP, this change can be interpreted as a typical “stress leucogram”. In up to 65 % of cats with FIP, anemia is present with usually only a mild decrease in hematocrit. Thrombocytopenia can be commonly found, mainly as a result of DIC. In experimental infection, thrombocytopenia was detected as early as 4 days after infection.5 The most consistent laboratory finding in cats with FIP is an increase in total serum protein concentration. This is found in about 50 % of cats with effusion and 70 % of cats without effusion. This increase in total protein is caused by increased globulins, mainly γ-globulins, also leading to a decrease in the albumin to globulin ratio. In experimental infections, an early increase of α2-globulins was reported, while γ-globulins and antibody titers increase just before appearance of clinical signs. The characteristically high levels of γ-globulins and the increased antibody titers invite the conclusion that hypergammaglobulinemia is due to a specific anti-FCoV immune response. Antibody titers and hypergammaglobulinemia show a linear correlation, but the wide variation in anti-FCoV titers at a given concentration of γ-globulins indicates that additional (auto-immune) reactions occur during the pathogenesis of FIP.6,7 It has been discussed that additionally stimulation of B cells by interleukin-6, which is produced as part of the disease process, contributes to the increase in γ-globulins.8 Total protein in cats with FIP can reach very high concentrations of up to 12 g/dl (120 g/l) and more. This, however, reflects the chronic antigenic stimulation that in general can be caused by any chronic infection that the cat is not able to clear through its immune response. Even if the serum total protein concentration is ≥ 12 g/dl, the likelihood of FIP is only 90 %. Cats with these high serum protein concentrations that do not have FIP may suffer from severe chronic stomatitis (probably due to chronic antigen stimulation in calicivirus carriers), chronic upper respiratory disease, dirofilariosis, or multiple myeloma.4 In a recently performed study in cats with FIP, comparison of total serum protein concentration, γ-globulins, and albumin to globulin ratio revealed that the albumin to globulin ratio has a statistically significantly better diagnostic value than the other 2 parameters. An optimum cut-off value (maximum efficiency) of 0.8 was determined for the albumin to globulin ratio. If the serum albumin to globulin ratio is less than 0.8, the probability that the cat has FIP is high (positive predictive value 92 %); if the albumin to globulin ratio is higher than 0.8, the cat likely does not have FIP (negative predictive value 61 %).4
If there is effusion, the most important diagnostic step is to sample the fluid because tests on effusion have a much higher diagnostic value than tests that can be performed in blood. Only about half of the cats with effusions suffer from FIP.9 The effusion in FIP is usually classified as a modified transudate or exudate combining characteristics of both. The protein content is usually very high (> 3.5 g/dl), whereas the cellular content is low and approaches that of a pure transudate (< 5000 nucleated cells/ml). Other diseases causing similar effusion include lymphoma, heart failure, cholangiohepatitis, and bacterial peritonitis or pleuritis. Cytology of the effusion in cats with FIP typically shows a pyogranulomatous character with predominantly macrophages and neutrophils. Cytology may appear similar in cats with bacterial serositis or sometimes with lymphoma; these effusions often can be differentiated, however, by the presence of malignant cells or bacteria, respectively. A simple test, the so-called “Rivalta’s test”, has been used to differentiate transudates from exudates. This test seems to be very useful in cats to differentiate between effusions due to FIP and effusions caused by other diseases. It is not only the high protein content, but high concentrations of fibrin and inflammatory mediators that induce a positive reaction. To perform this test, 3 quarters of a reagent tube are filled with distilled water, to which 1 drop of acetic acid (98 %) is added and is mixed thoroughly. On the surface of this solution, 1 drop of the effusion fluid is carefully layered. If the drop disappears and the solution remains clear, the Rivalta’s test is defined as negative. If the drop retains its shape, stays attached to the surface or slowly floats down to the bottom of the tube (drop- or jelly-fish-like), the Rivalta’s test is defined as positive. The Rivalta’s test had a positive predictive value of 86 % and a negative predictive value of 97 %.4 There are some false-positive results in cats with bacterial peritonitis. Those effusions, however, are usually easy to differentiate (through macroscopic examination, cytology, and bacterial culture). Some cats with lymphoma may also react positively in the Rivalta’s test, but many of these can be differentiated cytologically.
Measurement of Antibodies
Antibody titers measured in serum are an extensively used diagnostic tool. However, in view of the facts that a large percentage of the healthy cat population is FCoV antibody-positive, that high and rising titers are frequently found in asymptomatic cats, and that most of those cats will never develop FIP, antibody titers must be interpreted extremely cautiously. Antibody testing has a certain role in the management of FIP when it is done by appropriate methodologies and results are properly interpreted. Antibody testing can only be useful if the laboratory is reliable and consistent. Methodologies and antibody titer results may vary significantly. A single serum sample, divided and sent to 5 different laboratories yielded 5 different results . The laboratory should have established 2 levels to enable useful interpretation; one is its least significant level of reactivity (or lowest positive titer) and the other is its highest antibody titer value.
Presence of antibodies does not indicate FIP; absence of antibodies does not exclude FIP. Low or medium titers do not have any diagnostic value. Approximately 10 % of the cats with clinically manifest FIP have negative results.4 This is either because large amounts of virus in the cat's body bind to antibodies and render them unavailable to bind antigen in the antibody test or because the antibodies are lost into the effusion when protein is translocated in vasculitis. Of a certain diagnostic value are very high titers. If the highest measurable titer is present, this finding increases the likelihood of FIP. In a recent study, the probability of FIP was 94 % in cats with the highest titer when investigating a cat population in which FIP was suspected.4 The height of the antibody titer directly correlates with the amount of virus that is shed with feces; cats with high antibody titers are more likely to shed FCoV and shed more consistently with higher amounts of the virus. Thus, height of the titer is directly correlated with the virus replication rate and the amount of virus in the intestines.
Some studies evaluated the diagnostic value of antibody detection in fluids other than the serum, such as in effusions or CSF. Presence of antibodies in effusion is correlated with the presence of antibodies in blood, and thus, its diagnostic value is also limited.4,10 Some authors determined the diagnostic value of antibody detection in CSF and found a very good correlation to the presence of FIP when compared to histopathology, while in other studies, there was no significant difference in antibody titers in CSF from cats with neurologic signs due to FIP compared to cats with other neurologic diseases confirmed by histopathology.11
PCR
Reverse-transcription polymerase chain reaction (RT-PCR) can be performed to reverse-transcribe coronavirus RNA to cDNA and then make visually detectable large quantities of DNA. Although FIP-causing viruses are genetic mutants of harmless enteric FCoV, numerous sites exist in the 3C and 7B genes that can be mutated or deleted and confer on the virus the capability to infect and replicate within macrophages. Sometimes the change can be a single RNA base. As a result, PCR primers to discriminate between FIP-causing viruses and harmless enteric FCoV can not be designed and it is not possible to distinguish between a mutated and a non-mutated virus by PCR.12 There are a number of reasons why RT-PCR results are not always easy to interpret. False negative PCR results are common. The assay requires reverse transcription of viral RNA to DNA prior to amplification of DNA, and degradation of RNA could be a potential problem since RNases are virtually ubiquitous. There may be sufficient strain and nucleotide sequence variation such that the target sequence chosen for this assay may not detect all virus strains.13 There are also a number of reasons for false positive results. First, the assay does not distinguish between “virulent” and “avirulent” FCoV strains, nor will it discriminate FCoV from canine coronavirus (CCV) and transmissible gastroenteritis virus (TGEV). Second, recent studies support the hypothesis that viremia occurs not only in cats with FIP but also in healthy carriers. FCoV RNA could be detected in the blood of cats with FIP but also in healthy cats that did not develop FIP for a period of up to 70 months. In one study it was shown that in households, in which FCoV is endemic, up to 80 % of the cats can be viremic (PCR positive), irrespective of their health status, and that the presence of viremia does not appear to predispose the cats to the development of FIP.14
Antigen Detection
Other methods to detect the virus itself include searching for the presence of FCoV antigen. In a recent study involving a large number of cats, immunofluorescence staining of intracellular FCoV antigen in macrophages of the effusion had a positive predictive value of 100 %. There were no false positive results. Unfortunately, the negative predictive value was not very high (57 %). Cases that stained negative (although the cats did have FIP) can be explained by the fact that the number of macrophages on the effusion smear is sometimes insufficient. Another explanation is a potential masking of the antigen by competitive binding of FCoV antibodies in the effusion that displace binding of fluorescence antibodies.4 Immunohistochemistry can also be used to detect the expression of FCoV antigen in tissue.15 In one study, immunohistochemistry was used to detect intracellular FCoV antigen in paraffin-embedded tissues of euthanised cats; FCoV antigen only was found in macrophages of cats that had FIP and not in control cats.16 In another study, FCoV antigen was demonstrated in the membrana nicticans of cats with FIP.17 Immunostaining cannot differentiate between the “harmless” non-mutated FCoV and the mutated FIP-causing FCoV. But obviously, only FIP-causing virus is able to replicate in sufficiently large amounts in macrophages that it results in a positive staining. Therefore, besides histopathology (if pathognomonic lesions are present) detection of intracellular FCoV antigen by immunofluorescence or immunohistochemistry is the only way to definitively diagnose FIP. This tool should be used whenever possible.
Therapy
Treatment for FIP is almost invariably doomed to failure because cats with clinical FIP eventually die. However, some cats with milder clinical signs may survive for several months and enjoy some quality of life with treatment. Once clinical signs become debilitating and weight and appetite decline, however, the owner must be prepared for the reality that their cat is dying. As FIP is an immune-mediated disease, treatment is aimed at controlling the immune response to FCoV, and the most successful treatments consist of relatively high doses of immunosuppressive and anti-inflammatory drugs. Immune suppressive drugs such as prednisone (2 to 4 mg/kg every 24 hours orally) or cyclophosphamide (2 to 4 mg/kg 4 times a week orally) may slow disease progression but do not produce a cure. A thromboxane synthetase inhibitor (ozagrel hydrochloride) which inhibits platelet aggregation has been used in a few cats and has lead to some improvement of clinical signs.18
Ribavirin, 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, RTCA, is a nucleoside analog but in contrast to the most common antiviral compounds that act primarily to inhibit reverse transcriptase activity by causing premature chain termination during the transcription of DNA from the single-stranded RNA template, ribavirin allows DNA synthesis to occur, but prevents the formation of viral proteins, most likely by interfering with capping of viral mRNA. In vivo, therapeutic concentrations are difficult to achieve due to toxicity, and cats are extremely sensitive to the side effects (e.g. hemolysis). Although active against FCoV in vitro, ribavirin was not effective to treat cats with FIP. In one study, ribavirin was administered (16.5 mg/kg every 24 hours for 10 to 14 days orally, intramuscularly, or intravenously) to specific pathogen-free kittens 18 hours after experimental challenge exposure with a FIP-causing virus. All kittens, including ribavirin-treated and untreated kittens, succumbed to FIP. Clinical signs of disease were even more severe in the ribavirin-treated kittens and their mean survival times were shortened. The authors tried to decrease the toxicity of ribavirin by incorporating it into lecithin-containing liposomes and giving it at lower dosage (5 mg/kg) intravenously to cats challenged with a FIP-causing virus. They were, however, not able to reach a therapeutic concentration with this regimen.19
Human interferon-α, IFN-α, has immunomodulatory and antiviral activity and is active against many DNA and RNA viruses including FCoV. In vitro, antiviral activity of human interferon-α against a FIP-causing FCoV strain was demonstrated. Combination of interferon-α with ribavirin in vitro resulted in antiviral effects significantly greater than the sum of the observed effects from either ribavirin or interferon-α alone, indicating synergistic interactions. Human interferon-α treatment was used in 74 cats (52 treated, 22 controls) with experimentally induced FIP that received interferon-α, Propionibacterium acnes, a combination, or placebo. The prophylactic and therapeutic administration of high doses (104 or 106 IU/kg) interferon-α did not significantly reduce the mortality in treated versus untreated cats; only in cats treated with 106 IU/kg interferon-α and Propionibacterium acnes the mean survival time was significantly prolonged, but only for a few days.20
Recently, the corresponding feline interferon, feline interferon-ω, was licensed for use in veterinary medicine in some European countries and Japan. Interferons are species-specific and the feline interferon differs from the human one not only concerning its antigenicity (therefore causing no antibody development in cats) but also concerning its antiviral efficacy in feline cells. Feline interferon-ω is a recombinant product which is produced by baculoviruses containing the feline sequence for this interferon that replicate in silkworms after infection; subsequently, feline interferon-ω is purified out of homogenized silkworm preparation. FCoV replication is inhibited by feline interferon-ω in vitro. In one study (not controlled and only including a small number of cats with FIP not confirmed), 12 cats that were suspected to have FIP were treated with interferon-ω in combination with glucocorticoids and supportive care. Although most cats died, 4 cats survived over a period of 2 years; all of them had initially presented with effusions.21 In a recently performed randomized placebo-controlled double-blind treatment trial, 34 cats with FIP were treated with interferon-ω or placebo. In all cats, FIP was confirmed by histology and/or immunohistochemical or immunofluorescence staining of FCoV antigen in effusion or tissue macrophages. All cats received glucocorticoids, either as dexamethasone in case of effusion (1 mg/kg intrathoracic or intraperitoneal injection every 24 hours) or prednisolone (2 mg/kg orally every 24 hours). In addition, cats received either placebo or interferon-ω at 106 U/kg subcutaneously every 24 hours for 7 days and subsequently once every week. There was no statistically significant difference in the mean survival time of cats treated with interferon-ω or placebo. Cats survived for a period of 3 to 200 days before they had to be euthanised with a mean survival time of 17 days. There was only one long term surviver (> 3 months) who appeared to be in the interferon-ω group. This cat had been presented with effusion that totally disappeared during the treatment period. It did not show any signs of disease until it was presented with severe upper respiratory signs and a FIP relapse 200 days after treatment initiation and had to be euthanised due to its bad clinical condition.
References
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New Aspects of Diagnosis and Treatment
Monday, October 1, 2007
