Summary of Section 7: Diagnosis of FIP; Section 7.5: Direct Detection of FCoV FCoV antigen detection by immunostaining  Immunostaining exploits the binding of antibodies to host-cell-associated FCoV antigens, which are subsequently visualised by enzymatic or immunofluorescent reactions producing a colour change in a process called immunohistochemistry (IHC) on biopsies or immunocytochemistry (ICC) or immunofluorescence (IF) on cytology samples (such as effusion and fine-needle aspirate [FNA] sample smears).  The histopathological and cytological changes associated with FIP are typically pyogranulomatous.  Definitive diagnosis of FIP relies on consistent histopathological changes in affected tissues in addition to FCoV antigen immunostaining by IHC.  Consistent cytological changes in affected tissues in addition to FCoV antigen immunostaining by ICC or IF is also highly supportive of a diagnosis. Although positive FCoV antigen immunostaining can usually be used to confirm the diagnosis, a negative result does not exclude FIP as FCoV antigens can be variably distributed within lesions and might not be detected in all samples prepared from FIP-affected tissues or samples (e.g., if an effusion is cell-poor and/or the FCoV antigen is masked by FCoV antibodies in the effusion). It is important for clinicians to be aware of variations in immunostaining techniques and to be familiar with the specificity of the methodology employed by their local laboratory, as well as confirmation of the inclusion of negative controls in testing, when interpreting positive results.  Differential diagnoses for pyogranulomatous inflammation include other infections (mycobacteria, toxoplasmosis, actinomyces, nocardia, rhodococcus, bartonella, pseudomonas and fungi) as well as idiopathic sterile pyogranulomatous disease.  The sample sites most likely to be useful are those that are affected by the FIP disease, and inference of this can be gained from the clinical signs as well as results of diagnostic testing (e.g., ascites, neurological signs, imaging results, pyogranulomatous inflammation on FNA cytology). Biopsy samples of affected tissues (e.g., liver, kidney, spleen, mesenteric lymph nodes) can be collected by laparotomy, laparoscopy or ultrasound-guided tru-cut for histopathology and immunostaining, whereas effusions, FNAs (e.g., of mesenteric lymph nodes), cerebrospinal fluid (CSF) and aqueous humour samples can be collected for cytology and immunostaining. It is wise to consult the diagnostic laboratory before submitting samples for ICC or IF as their preferences for how samples should be prepared before sending vary. FCoV RNA detection by reverse-transcriptase polymerase chain reaction (RT-PCR)  FCoV RT-PCR assays can be used to detect FCoV RNA in blood, effusion, tissue (including samples obtained by FNAs), CSF, or aqueous humour samples. The RT-PCR assays used should be quantitative and report the FCoV load (amount) present in the analysed sample. The load is helpful because the systemic FCoV infection that can occur in healthy cats and cats without FIP have lower FCoV viral loads than in cats with FIP. Thus, a positive FCoV RT-PCR result on a sample is not totally specific for FIP, but positive results with a high FCoV load on samples from cats with signs consistent with FIP are very supportive of a diagnosis of FIP, and often this is adequate evidence upon which to start a cat with antiviral FIP treatment. However, a negative result cannot rule out a diagnosis of FIP since the levels of FCoV in samples can be too low or have too variable a distribution (and thus not present in the sample analysed) to be detectable by PCR. It is wise to consult the diagnostic laboratory before submitting samples for RT-PCR, as their preferences for how samples should be prepared before sending vary (e.g., centrifugation of effusions, preservation advice).  Recent studies using RT-PCR on blood samples have shown more promising results than previously, with high levels of FCoV RNA detectable, suggesting that blood samples could be revisited as a diagnostic sample to support a diagnosis of FIP.  RT-PCR analysis of effusion samples in cats with FIP is often positive (72–100% of samples) for FCoV RNA, and cats without FIP are usually RT-PCR-negative, and the presence of FCoV RNA, particularly in high levels, in an effusion that also has cytological and biochemical features suggestive of FIP, is highly supportive of a diagnosis of FIP.  Whilst tissue biopsy samples obtained from affected tissues in cats with FIP usually show high levels of FCoV RNA in them, as determined by RT-PCR, such samples, if collected, should ideally be submitted for histopathology and IHC, as this allows for a definitive diagnosis of FIP.  FNAs are a good sample type for FCoV RT-PCR, with the advantage of relatively easy collection. The sample site should be guided by where pathology is likely based on clinical signs and other diagnostic investigations, but promising results on FNAs collected from mesenteric lymph nodes from cats with FIP that did not have effusions have been obtained.  CSF and aqueous humour FCoV RT-PCR in cats with neurological signs or ocular signs, respectively, can also be helpful.  RT-PCR on faecal samples is only useful to identify cats shedding FCoV for the management of FCoV in multi-cat households. Faecal RT-PCR is not useful for the diagnosis of FIP as many healthy cats without FIP shed FCoV. Characterising FCoV spike (S)-gene mutations following positive RT-PCR for FCoV RNA  Following the detection of FCoV RNA in a sample by RT-PCR, varied molecular techniques (e.g., pyrosequencing and Sanger sequencing often used in research, or methods that detect and quantify specific FCoV mutation sequences, such as the commercially available allelic discrimination assay) can be used to derive S-gene sequence data for the FCoV present. Such techniques are only successful at determining the FCoV sequence present when high loads of FCoV RNA are present, so successful S-gene-mutation analysis at least suggests that the sample contained high levels of FCoV RNA, which is highly supportive of a diagnosis of FIP. However, research has shown great variability in results when detecting S-gene mutations using the different methods, making it difficult to rely on S-gene-mutation analysis as being confirmative for FIP, especially when the commercially available allelic discrimination assay is used.