Where we are with understanding the cause of FIP? Feline coronavirus (FCoV) infection is very common in cats. Infections with FCoV are usually asymptomatic but result in FIP in around 5–10% of cats1. Asymptomatic FCoV infection was previously believed to be confined to the intestinal tract, but healthy FCoV-infected cats can have systemic FCoV infection, albeit with lower viral loads than cats with FIP2-4. Why FCoVs result in FIP in some cats and not in the majority of FCoV infected cats is not clear. Viral factors are important. FCoVs have a transmembrane spike (S) protein that binds to the host (feline) receptor, mediating host cell entry. Mutations in the S gene can result in amino acid substitutions in the transcribed S protein that influence the tropism of FCoV, and these are believed to be associated with the ability of FCoV replication to occur outside of the intestinal tract (monocyte/macrophage tropism) as systemic FCoV infection5, which is a prerequisite to the development of FIP. Other viral factors are also likely to be important following systemic FCoV infection6. Host factors are also very likely to play an important part in FIP development. Can I make a definitive diagnosis of FIP in A suspected case?
This requires histopathological examination of tissues with detection of FCoV antigen within lesions by immunohistochemistry. Immunostaining of FCoV antigen in effusion samples showing biochemical and cytological features consistent with FIP is also likely to be adequate to definitively diagnose FIP in effusive cases.
- Evaluating any background evidence for FIP
- Are the clinical signs consistent with FIP?
- Are haematology and biochemistry consistent with FIP?
- Is an effusion present to sample?
These are important to consider and are described in detail in one of the author’s publications7.
FCoV Reverse-Transcriptase (RT)-PCR
RT-quantitative (q)PCR assays detect and quantify FCoV RNA. These assays detect any FCoV RNA and so are not specific for FIP-associated FCoVs and cannot be used to definitively diagnose FIP as both cats with and without FIP can show positive RT-PCR results. FCoV RT-PCR can be used to detect FCoV RNA in tissue (biopsy or [ultrasound-guided] fine needle aspirates [FNAs]), effusion, CSF or aqueous humour samples from suspected cases of FIP.
Selection of appropriate samples to submit for RT-PCR can be guided by clinical signs such as presence of effusions, ocular/neurological signs, imaging, cytology and non-invasive sampling methods are generally preferred, particularly in sick cats. Tissue samples from cats with FIP are significantly more likely to be FCoV RT-PCR positive8,9, and have significantly higher FCoV loads by RT-qPCR5, than tissues samples from cats without FIP, although cats without FIP can still be positive for FCoV by RT-PCR in tissues. A recent extensive study evaluating FCoV RT-PCR in 260 tissue samples from 57 cats with FIP, and 258 tissue samples from 45 cats without FIP8, 90.4% of tissue samples from cats with FIP were FCoV RT-qPCR positive compared to only 7.8% of tissue samples from cats without FIP. In cats with FIP, Thus, the presence of high levels of FCoV RNA in tissue samples is highly supportive of a diagnosis of FIP. Effusion samples in FIP cats often contain FCoV RNA10, which can be detected by RT-PCR. Published studies have amplified FCoV RNA in most (72–100%) effusion samples from cats with FIP9,11-13 but usually not in any non-FIP effusion samples11-13. However recent studies have challenged specificity; one study8 amplified FCoV RNA, albeit at a low level, in abdominal fluid from one (out of 29) control cats that did not have FIP and another14 amplified FCoV from 3 (2 of these had low levels of FCoV) of 24 control non-FIP cat that had effusions tested. Lastly a recent study9 amplified FCoV (FCoV levels not reported) from the effusion of one cat with an intestinal carcino ma (out of 6 control cats with effusions tested). Despite this, the presence of FCoV RNA, particularly moderate to high levels, in an effusion that also has cytological and biochemical features consistent with FIP, is highly supportive of a diagnosis of FIP. Successful RT-PCR is possible on MLN FNAs in cats with FIP15; 18 of 20 cats with non-effusive FIP were positive for FCoV by RT-qPCR with the remaining 2 cats giving negative results, although these presented primarily with neurological signs. Of 26 non-FIP cats, 25 showed negative FCoV RT-qPCR results. In this study, the sensitivity of MLN FNA FCoV RT-qPCR was 90% and specificity 96.1%. Not all cats in this study were confirmed as having FIP using histopathology with or without immunohistochemistry and samples were often collect post-mortem. Nevertheless, the study suggests that RT-PCR on MLN FNAs collected from cats with (non-effusive) FIP could be useful to support a diagnosis of FIP.
FCoV RT-PCR Followed by S Gene Mutation Analysis
Following the detection of FCoV RNA in a sample by RT-PCR, it may be possible to then characterise targeted sequences of the FCoV genome, especially the S gene, using molecular techniques. Such sequence characterisation would be extremely useful if FIP-specific mutations existed.
Although research16,17 documented so-called FIP-specific S gene mutations, these were identified by comparing the sequences of FCoVs found in the tissues of FIP cats with those found in the faeces of healthy non-FIP cats. We hypothesized that these sequence mutations could reflect systemic FCoV (i.e., monocyte/macrophage-associated FCoV compared to intestinal epithelium-associated FCoV) rather than being specific for FIP, knowing that non-FIP cats can have systemic FCoV infection. We therefore compared the S gene sequences of FCoV detected in the tissues of FIP cats with those detected in the tissues of non-FIP cats5. This allowed us to evaluate the S gene sequences of FCoVs associated with systemic FCoV infection in both non-FIP and FIP cases. We found that the S gene mutations present in most of the FIP tissues were also present in most of the tissues of non-FIP cats that had systemic FCoV infection. A recent more extensive study confirmed the same findings8, and calculated that if the identification of S gene mutated FCoVs was included as an additional confirmatory step to the detection of FCoV by RT-PCR alone, this only slightly increased specificity for the diagnosis of FIP in tissue samples (from 92.6% for FCoV RT-PCR alone to 94.6% with the addition of S gene mutation analysis) but moderately decreased sensitivity (from 89.8% to 80.9%, respectively, mainly because mutation analysis was not possible in all tissue samples). Other studies on mutation analysis11,14,18 have reported lower sensitivities but higher specificities compared to FCoV RT-PCR alone than those in our studies. One should remember that if a mutation assay is heavily reliant on having a significant FCoV load in the sample to enable sequencing, its sensitivity can appear to be quite poor as some samples from FIP cats may not have adequate FCoV loads on which to perform successful sequencing. Conversely, such assays may appear to have good specificity as they are unable to sequence mutated sequences in cats without FIP, and thus don’t generate false positives. Hence significant increases in specificity for mutation assays over FCoV RT-PCR alone may be due to their inability to identify mutated FCoV in cats without FIP.
Immunostaining for FCoV Antigen Should Be Performed If Possible
Immunostaining is performed on formalin-fixed tissues using immunohistochemistry (IHC) or on cytological (typically effusion) samples using immunocytochemistry (ICC) or immunofluorescence. Positive FCoV antigen immunostaining of tissue biopsies is said to confirm a diagnosis of FIP (i.e., it is very specific) but a negative result does not exclude FIP as a diagnosis as FCoV antigens may be variably distributed within lesions19 and thus are not detected in all histopathological sections prepared from lesions from FIP cases6.
This may be overcome by taking multiple and/or large samples with confirmed pathology, as well as possibly requesting additional sections of biopsies with pathology to be cut and stained. Immunostaining of effusion samples has shown variable sensitivity (57–100%)20-22. Since this technique relies on staining FCoV within macrophages in the effusion, and the effusion is often cell-poor and/or the FCoV antigen is masked by FCoV antibodies in the effusion, a false negative result may be obtained. Effusion immunostaining is thought to be very specific; recent studies questioning specificity20,21 may be due to the methodology used causing non-specific staining. The use of immunostaining has recently been described in MLN FNA samples obtained at post-mortem examination from 41 cats with suspected FIP23. FIP was confirmed in 30 cats by histopathology and positive IHC whilst 11 cats were confirmed as having diseases other than FIP by histopathology and negative IHC, although MLNs were not necessarily evaluated. Of the 30 cats with FIP, 17 (53%) were MLN FNA ICC positive, and of 11 cats without FIP, 1 (9%) was MLN FNA ICC positive; thus, ICC had a sensitivity of 53% and a specificity of 91%. The ICC technique described23 could have been insensitive due to the antibody type used and degradation of FCoV and/or cells due to the post-mortem collection. Further evaluation of ICC on MLN FNA samples collected ante-mortem from cats is required.
References are available upon request.