Diagnosis
Diagnosis of FIP intra vitam is extremely challenging. In addition, a definitive diagnosis may not always be possible, e.g., because of the invasiveness of biopsies in a sick cat. Difficulties in definitively diagnosing FIP arise from an absence of non-invasive confirmatory tests in cats with no effusion. Presence of effusion should first be ruled out because obtaining effusion and analysis is very useful and relatively non-invasive. In cats with no effusion, several parameters, including the background of the cat, history, presence of clinical signs, laboratory changes, and antibody titres [Rohrer et al, 1993] should be used to help to inform the decision about appropriate further diagnostic procedures.
Haematology
Haematology results are often altered in cats with FIP, but the changes are not pathognomonic. White blood cell counts can be decreased or increased. Lymphopenia is commonly present; however, lymphopenia in combination with neutrophilia is generally common in cats as a typical “stress leukogram” and can occur in many other diseases. However, a normal lymphocyte count makes FIP less likely. A mild to moderate non-regenerative anaemia is also a common, but non-specific, finding, which may occur in almost any chronic disease of the cat.
A very common laboratory finding in cats with FIP is an increase in total serum protein concentration caused by a rise in globulins, mainly γ-globulins [Paltrinieri et al, 2001; 2002]. In one study hyperglobulinaemia was found in about 50% of cats with effusion and 70% of cats without effusion [Sparkes et al, 1994]. Following experimental infection, an early increase of α2-globulins is seen, while γ-globulins and antibody titres increase just prior to the onset of clinical signs [Pedersen 1995; Gunn-Moore et al, 1998]. Serum total protein levels in cats with FIP can reach very high concentrations of up to 120 g/l (12 g/dl) or higher. In some studies, the albumin to globulin ratio was found to have a significantly higher diagnostic value than either total serum protein or γ-globulin concentrations, because a decrease in serum albumin also may occur through a decrease in production [Shelly et al, 1988; Rohrer et al, 1993; Hartmann et al, 2003]. Low albumin is usually associated with protein loss caused by glomerulopathy secondary to immune complex deposition or by extravasation of protein-rich fluid during vasculitis [Hartmann et al, 2003]. An optimum cut-off value (maximum efficiency) of 0.8 was determined for the albumin to globulin ratio [Hartmann et al, 2003]. Serum protein electrophoresis may show both polyclonal and monoclonal hypergammaglobulinaemia as well as increases in acute phase proteins. Other laboratory parameters (liver enzymes, bilirubin, urea, creatinine) can be variably elevated depending on the degree and localization of organ damage, but are generally not helpful in establishing an etiological diagnosis. Hyperbilirubinemia and icterus are often observed and frequently are a reflection of hepatic necrosis [Hartmann et al, 2003]. Sometimes, bilirubin is increased without evidence of haemolysis, liver disease, or cholestasis; this unusual change is otherwise only observed in septic animals. Bilirubin metabolism and excretion into the biliary system is compromised in these cats due to high levels of TNF-α that inhibit transmembrane transport. Thus, high bilirubin in the absence of haemolysis and elevation of liver enzyme activity should raise the suspicion of FIP. Recent research has focused on the diagnostic value of acute phase reaction parameters including α1-acid glycoprotein (AGP), a serum acute phase protein that is elevated in cats with FIP [Duthie et al, 1997; Paltrinieri, 2008]. High serum AGP levels (>3 mg/ml) can support the diagnosis of FIP [Paltrinieri et al, 2007a], but levels also rise in other inflammatory conditions and thus, these changes are not specific. Additionally, AGP may also be high in asymptomatic cats infected with FCoV, especially in households where infection is endemic [Paltrinieri et al, 2007a].
Tests on effusion fluid
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 on blood. Only about half of the cats with effusion suffer from FIP [Hirschberger et al, 1995]. Although effusions of clear yellow colour and sticky consistency are often called “typical”, the presence of this type of fluid in body cavities alone is not diagnostic. Sometimes the fluid has a totally different appearance and some cases of FIP with pure chylous effusion have been reported [Savary et al, 2001]. Usually the protein content is very high (>35g/dl) and consistent with an exudate, whereas the cellular content is low (< 5000 nucleated cells/ml) and approaches that of a modified transudate or pure transudate. Cytology of the effusion in cats with FIP shows a variable picture but often consists predominantly of macrophages and neutrophils. Electrophoresis in effusions is a diagnostic tool with a high positive predictive value if albumin/globulin ratio is < 0.4 and a high negative predictive value if the ratio is > 0.8 [Shelly et al, 1988]. Major differential diagnoses of cats with similar effusions include inflammatory liver disease, lymphoma, heart failure, and bacterial peritonitis or pleuritis.
“Rivalta’s test” is a very simple, inexpensive method that does not require special laboratory equipment and can be easily performed in private practice. This test was originally developed by the Italian researcher Rivalta around 1900 and was used to differentiate transudates and exudates in human patients. This test is very useful in cats to differentiate between effusions due to FIP and effusions caused by other diseases [Hartmann et al, 2003]. Not only the high protein content, but high concentrations of fibrinogen and inflammatory mediators lead to a positive reaction.
Box 1. Rivalta’s test
To perform this test, a transparent reagent tube (volume 10 ml) is filled with approximately 7-8 ml distilled water, to which 1 drop of acetic acid (98%) is added and 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 high positive predictive value (86%) and a very high negative predictive value for FIP (96%) in a study in which cats that presented with effusion were investigated (prevalence of FIP 51%) [Hartmann et al, 2003]. Positive Rivalta’s test results can occur in cats with bacterial peritonitis or lymphoma. Those effusions, however, are usually easy to differentiate through macroscopic examination, cytology, and/or bacterial culture.
Cerebrospinal Fluid
Analysis of cerebrospinal fluid (CSF) from cats with neurological signs due to FIP lesions may reveal elevated protein (50 - 350 mg/dl with a normal value of less than 25 mg/dl) and pleocytosis (100 - 10000 nucleated cells/ml) containing mainly neutrophils, lymphocytes, and macrophages [Li et al, 1994; Rand et al, 1994; Foley et al, 2003], which is, however, a relatively non-specific finding. Many cats with neurological signs caused by FIP have normal CSF.
Antibodies
Antibody titres measured in serum can contribute to the diagnoses if interpreted with care. A high percentage of healthy cats are FCoV antibody-positive and most of those cats will never develop FIP. Thus, antibody titres must be interpreted with extreme caution; it has been contended that more cats have died of false interpretation of FCoV antibody test results than of FIP [Pedersen, 1995a]. There is no “FIP antibody test”, all that can be measured is antibodies again FCoV. Methodology (and thus, antibody titre results) may vary significantly between laboratories. It is important to realize that the presence of antibodies does not indicate FIP and absence of antibodies does not exclude FIP. Low or medium titres do not rule out FIP and approximately 10% of the cats with clinically manifest FIP have negative results [Hartmann et al, 2003]. In cats with fulminant FIP, titres may decrease terminally [Pedersen, 1995a]. 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 tests or because the antibodies are lost into the effusion when protein is extravasated in vasculitis. Very high titres can be of certain diagnostic value and increase the likelihood of FIP [Hartmann et al, 2003].
Measuring antibodies in fluids other than blood has been investigated [Boettcher et al, 2007; Foley et al, 1998]. However, interpretation of titres in effusion and cerebrospinal fluid is even more complicated than titres in blood and measurement of antibodies there is therefore not recommended.
FCoV Reverse-transcriptase polymerase chain reaction (RT-PCR)
FCoV RT-PCR in blood is sometimes used as diagnostic tool for the diagnosis of FIP. At the time of writing no PCR has been developed which can definitively diagnose FIP, and FCoV RT-PCR in blood is not recommended for the diagnosis of FIP. This is because it is not possible to distinguish between the FIP-inducing mutant and the non-mutated FCoV [Fehr et al, 1996]. Furthermore, positive FCoV RT-PCR results occur not only in cats with FIP but also in healthy carriers that did not develop FIP for a period of up to 70 months [Gunn-Moore et al, 1998b; Meli et al, 2004; Gamble et al, 1997; Herrewegh et al, 1997], and negative FCoV RT-PCR also occurs very commonly in cats with FIP [Hartmann et al, 2003]. Another approach is to measure messenger RNA by RT-PCR in blood with the rationale that levels of messenger RNA may correlate with the level of replication of FCoV and thus, be correlated with the presence of FIP. However, validity is unclear at present, 5 to 50 % of healthy were positive in that test [Simons et al, 2005; Can-Sahnak et al, 2007], and so far, the test is not available in Europe.
PCR in effusion or CSF has also been discussed as a diagnostic tool. However, data on the value of these approaches are not yet available.
Immunostaining of FCoV antigen in macrophages
Methods to detect the virus itself include the search for the presence of FCoV antigen in macrophages using immunofluorescence (in effusion macrophages) or immunohistochemistry (in tissue macrophages). While FCoV may be present systemically in cats without FIP, only in FIP will there be sufficiently large amounts of virus in macrophages to obtain positive staining. In a recent study in which a large number of cats with confirmed FIP and controls with other (confirmed) diseases were investigated (n = 171, prevalence of FIP 64%), positive immunofluorescence staining of intracellular FCoV antigen in macrophages of the effusion was 100 % predictive of FIP [Hartmann et al, 2003]. Unfortunately, the negative predictive value is not very high (57%), which can mainly be explained by low numbers of macrophages on effusion smears (even though cats have FIP) resulting in negative staining [Hartmann et al, 2003]. Immunohistochemistry can be used to detect the expression of FCoV antigen in tissue, and it also proved to be 100% predictive of FIP if positive [Tammer et al, 1995; Kipar et al 1998b]. However, invasive methods (e.g. laparotomy or laparoscopy) are usually necessary to obtain appropriate tissue samples. When the diagnostic sensitivity between true-cut biopsy (TCB) and fine-needle aspiration (FNA) of liver and kidney tissue obtained at necropsy was compared, the sensitivity of FNA was similar to TCB, but a higher sensitivity in the liver versus kidneys was observed [Giordano et al, 2005]. The value of ultrasound-guided FNA to diagnose FIP in vivo, however, still has to be investigated.
Therefore, there are 2 diagnostic strategies to obtain a definitive diagnosis of FIP. If there is effusion, immunofluorescence staining of FCoV antigen in effusion macrophages can diagnose FIP. If there is no effusion, tissue samples of affected organs have to be obtained. Histology is confirmative or immunohistochemical staining of FCoV antigen in tissue macrophages can be used to diagnose FIP. A diagnostic algorithm is provided in figure 7-1.
Figure 7-1. 