Winn’s Cat Health News blog has posted numerous summaries about feline coronavirus (FCoV) infection in cats and its more lethal form, feline infectious peritonitis (FIP). Feline coronavirus infection is very commonly found in cats and their environment, especially the multi-cat setting. Between 5 and 10% of FCoV infections will result in the fatal form, FIP.
Feline coronavirus is a RNA virus that undergoes a high rate of mutation during replication, resulting in clusters of genetically diverse species, or also known as quasispecies. This ability for genetic diversity, along with being able to recombine with other coronavirus strains, is the basis for their pathogenicity and cross-species transmission. There are two recognized serotypes: Type 1, which represents most of the field strains in naturally infected cats, and Type 2, which resulted from recombination between Type 1 FCoV and CCoV (canine coronavirus). It is through the genetic and serological differences in their transmembrane spike (S) gene and protein that creates this differentiating factor. The S protein has been found to be part of the virus that binds to the host receptor, facilitating host cell entry.
There are three major factors contributing to the development of FIP:
1) Viral factors – mutations in the fusion peptide sequence of the FCoV S gene and changes in the related furin cleavage site along with other mutations where FCoV develops its tropism for monocyte/macrophage cells and allows for systemic viral spread outside the intestinal tract and leading to development of FIP
2) Host factors – these include the host immune response (such as the significant decrease of T-lymphocytes with FIP), monocyte ability to sustain virus replication, breed and genetics
3) Environmental factors – this includes stress levels and overcrowding in housing that might increase viral replication in individual cats, therefore increasing viral mutations which can lead to FIP development
The Road to FIP Diagnosis
Often prior to the step of determining a definitive diagnosis of FIP, veterinarians will develop a disease differential list where FIP is high or first on the list of potential diagnoses. This often is due to the signalment and history of the patient, clinical signs and results of a thorough examination and routine clinicopathological test results. Experience of the practitioner in diagnosing and managing many cases of FIP can also aid in increasing the degree of suspicion of FIP. A number of diseases to consider in the differential diagnosis of FIP are: toxoplasmosis, lymphocytic cholangitis, neoplasia (e.g. lymphoma, abdominal carcinoma), pancreatitis, retroviral infection, mycobacterial infection (including tuberculosis), pyothorax, sepsis, septic peritonitis, congestive heart failure, rabies (neurologic), and others.
Signalment and history: young cats (less than 3 years of age and especially less than 2 years of age) and a different smaller peak of cases in cats older than 10 years of age. Male cats seem to have a slightly higher risk and some breeds in some countries. Frequently, there is a recent history of a stressful event – adoption, shelter exposure, neutering, vaccination, other infections like upper respiratory tract disease – could be a trigger for FIP development in a cat infected with FCoV. Living in a multi-cat environment may increase FCoV seropositivity, FIP can occur in small cat households too.
Clinical signs: due to vasculopathy causing effusions (wet), or granuloma formation (dry) lesions or a combination of the two. Up to 80% of FIP cases have effusions and most cases with effusions have visible granulomatous lesions on post-mortem examination. Signs include lethargy, anorexia, weight loss, or failure to thrive. In addition, there can be a fluctuating pyrexia that is non-responsive to drugs such as antibiotics or non-steroidal anti-inflammatories along with jaundice that is more commonly noted with effusive FIP. In cats with a history of pyrexia, one study found FIP was the most common differential diagnosis. Pyrexia was more significantly found in cases with effusive FIP than those with neurological non-effusive FIP.
Lymph node enlargement can be present in both effusive and non-effusive forms. Effusions can be present in the abdomen, pleural and pericardial spaces leading to dyspnea, tachypnea and/or abdominal distension. Non-effusive disease is typically neurological (focal, multifocal, or diffuse) and/or ocular signs (uveitis). In some cases, dermatological signs (papules or nodules) can be reported in dry FIP. With kidney involvement there may be renomegaly, and pneumonia with lung involvement. Focal lymph node enlargement or gastrointestinal disease may present as palpable abdominal mass(es). Repeated examinations may be required since clinical signs of FIP can change over time and the course of the disease.
Routine Laboratory Tests
Hematology – Changes in hematologic results in cats with FIP are often non-specific yet can be incorporated in building an increasing index of suspicion for a diagnosis. Lymphopenia is most commonly found (55-77% of cases are lymphopenic), neutrophilia (39-57%), left shift, and a mild to moderate normocytic, normochromic anemia (37-54%). Microcytosis (with or without anemia) has recently been associated with FIP.
Serum biochemistry – hyperglobulinemia: Hyperglobinemia is reported in 89% of cases, frequently with hypoalbuminemia or low-normal serum albumin concentrations. With lower albumin levels, hyperproteinemia may not always be present, especially in dry FIP cases. This combination of hyperglobulinemia and hypoalbuminemia also leads to a low albumin:globulin (A:G) ratio. Reports often suggest that an A:G ratio ratio of < 0.4 makes FIP very likely and an A:G ratio of 0.8 makes FIP very unlikely. The author states that she does not use a specific value but notes that the lower the A:G ratio value is, the greater the suspicion for a diagnosis of FIP, especially if other findings are consistent with FIP. An A:G ration of > 0.6 is useful to rule out but lower ratios do not help to rule in FIP.
Serum biochemistry – hyperbilirubinemia: Hyperbilirubinemia occurs in 21-63% of FIP cases. There are often no to mild increases in alanine transferase (ALT), alkaline phosphatase (ALP) or γ-glutamyltransferase activity. The presence of hyperbilirubinemia in the absence of elevated hepatic enzyme activities or severe anemia should increase the index of suspicion for FIP. Hyperbilirubinemia appears from reports to be identified more commonly just prior to death or euthanasia in these cases.
Serum biochemistry – acute phase proteins: α1-acid-glycoprotein (AGP) is an acute phase protein (APP). Elevations of AGP is not specific for FIP, marked elevations (> 1.5 mg/ml) are often seen with FIP. The magnitude of the increase can aid in the index of suspicion and diagnosis of FIP.
Feline coronavirus serology (FCoV): These serum antibody tests for FCoV are primarily ELISAs, indirect immunofluorescence antibody tests or others. A positive test indicates infection with FCoV and seroconversion (this occurs 2-3 weeks after infection). There is overlap in FCoV titers in FIP cats and non-FIP cats, so this test is of limited use in distinguishing cats with FIP. A positive result indicates exposure to FCoV only.
Effusion Sample Analysis: Because analysis of effusion samples from a suspected case of FIP is quite helpful in obtaining a diagnosis, getting a sample of any effusion should be a priority. Radiography is not considered as sensitive an imaging technique in obtaining samples compared to ultrasonography which can detect small volumes of fluid in body cavities such as the abdomen and thorax. Effusions associated with FIP are usually clear, viscous or sticky with straw-yellow color and a protein rich background on cytology, though chylous effusions are occasionally described. A total protein concentration of >35g/l (>50% globulins) are typical and FIP effusions frequently have low A:G ratios and raised AGP concentrations like those found in serum. These effusions also usually have poor cellular numbers (<5 x109 cells/l) which are pyogranulomatous in makeup with macrophages, non-degenerate neutrophils and few lymphocytes. Serology on effusions for FCoV antibodies is often not performed since results are often varied and non-diagnostic. Additional testing on effusion samples using immunostaining and RT-PCR will be discussed in the next part.
Rivalta test on effusion: 8 ml of distilled water is mixed with 1 drop of 98% acetic acid (or vinegar can be used). Then 1 drop of effusion is placed onto the surface of the liquid (usually in a test tube environment). A positive test will have the drop staying attached or connected with the surface or slowly floating to the bottom of the tube. A negative test is when the drop disappears and the solution remains clear. The test is subjective, therefore, the results are difficult to interpret properly as to whether a result is positive or negative.
Miscellaneous Tests: In cats with neurological signs of disease, imaging (MRI) can be performed and also procedures to collect cerebrospinal fluid (CSF) can be done with caution. Though some cases with FIP can demonstrate unremarkable CSF results, FIP cats can have marked elevations of protein (>2g/l) and an increased cell count (>8 x 106 cells/l) with the cell type predominantly neutrophils, mononuclear or mixed. Immunostaining and RT-PCR can also be done on CSF.
More Advanced Diagnostic Testing for FIP
RT-PCR: The author states that RT-PCR assays are available for the detection of FCoV, but they are not specific for FIP-associated FCoVs. It is advised that laboratories should be able to report the sensitivity and specificity of the RT-PCRs they use for detection of FCoV RNA. Immunostaining offers a definitive diagnosis where RT-PCR does not.
The samples from suspected cases of FIP that can be tested by RT-PCR for FCoV RNA are tissue, effusion, blood, CSF and aqueous humour. Tissue samples should not be formalin fixed. RT-PCR can be performed on fecal samples, yet this is used more to detect cats that are shedding virus in managing environments such as a multi-cat household. Cats with FIP are more likely to shed FCoV and have higher amounts of FCoV RNA in their feces than cats without FIP. An additional question is raised if further mutation analysis is useful, such as analyzing for fusion peptide sequence mutations. The authors found in one study that S gene mutations present in most of the FIP tissues they analyzed were also present in most tissues of non-FIP cats that had systemic FCoV infection. Another recent study confirmed these findings and found that including identification of the S gene mutated FCoVs as an additional confirmatory step only slight increased specificity and moderately decreased sensitivity of this test. Therefore, additional S-gene mutation analysis in FCoVs does not significantly improve the ability to diagnose FIP in fluid or effusion samples compared to RT-PCR alone.
Histopathology: Tissue samples are usually evaluated for the characteristic histopathological changes of FIP and when present, are regarded as reliable for diagnosis. Immunostaining for FCoV antigen is recommended to confirm the diagnosis. One study was mentioned that 5 of 8 FIP cases did not exhibit histopathology changes consistent with FIP even though large samples were taken. Diagnosis was then confirmed through positive FCoV antigen immunostaining.
Immunostaining of FCoV antigen: Immunostaining is used on formalin-fixed tissues using IHC, or cytology samples (usually effusions) using immunocytochemistry (ICC) or immunofluorescence (IF). Positive FCoV antigen immunostaining is very specific and confirms diagnosis. A negative result though does not exclude FIP as a diagnosis. Immunostaining of effusion samples has demonstrated variable sensitivity (due to the effusion is often cell or macrophage poor) so a false negative result may be obtained. Specificity of immunostaining of effusions can be higher though a recent study has raised questions about the specificity of ICC. More study needs to be done on application of ICC to CSF and aqueous humour samples to evaluate how useful this technique is for diagnosis in cases with neurological or ocular involvement.
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