Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis

Abstract

Objectives The aim of the study was to determine whether feline coronavirus (FCoV) RNA in effusion samples can be used as a diagnostic marker of feline infectious peritonitis (FIP); and in FCoV RNA-positive samples to examine amino acid codons in the FCoV spike protein at positions 1058 and 1060 where leucine and alanine, respectively, have been associated with systemic or virulent (FIP) FCoV infection. Methods Total RNA was extracted from effusion samples from 20 cats with confirmed FIP and 23 cats with other diseases. Feline coronavirus RNA was detected using a reverse transcriptase quantitative polymerase chain reaction assay (qRT-PCR), and positive samples underwent pyrosequencing of position 1058 with or without Sanger sequencing of position 1060 in the FCoV spike protein. Results Seventeen (85%) of the effusion samples from 20 cats with FIP were positive for FCoV RNA, whereas none of the 23 cats with other diseases were positive. Pyrosequencing of the 17 FCoV-positive samples showed that 11 (65%) of the cats had leucine and two (12%) had methionine at position 1058. Of the latter two samples with methionine, one had alanine at position 1060. Conclusions and relevance A positive FCoV qRT-PCR result on effusions appears specific for FIP and may be a useful diagnostic marker for FIP in cats with effusions. The majority of FCoVs contained amino acid changes previously associated with systemic spread or virulence (FIP) of the virus.

Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis

“…In total, 699 tissue, fluid and faeces samples were analysed from 102 cats, 57 with FIP and 45 without FIP. This is a marked increase in the number of samples analysed as compared to most previous studies [20,21,29,30], and contains similar numbers of effusion samples to two previous studies [19,31]. Some studies have examined the use of FCoV RT-PCR alone in the diagnosis of FIP using fluid samples [29,31].…”

“…Some studies have examined the use of FCoV RT-PCR alone in the diagnosis of FIP using fluid samples [29,31]. Other studies [19,21] compared FCoV RT-PCR to FCoV RT-PCR in combination with characterisation of S gene mutations in the diagnosis of FIP using body cavity fluids, these derived similar sensitivity (72 to 85% for FCoV RT-PCR alone and 60 to 64% for FCoV RT-PCR and S gene mutations characterisation) and specificity (100% for both FCoV RT-PCR alone and FCoV RT-PCR and S gene mutations characterisation) as obtained in this study (sensitivity 78.4% for FCoV RT-PCR alone and 60% FCoV RT-PCR and S gene mutations characterisation; specificity 97.9 and 97.9% respectively). As expected, in cats with histological changes consistent with FIP, individual tissues without FIP lesions were more likely to be FCoV RT-qPCR-negative or have lower copy numbers than those tissues in which FIP lesions were present.…”

“…Post-mortem tissue samples, faeces/faecal swab and body cavity fluid, if present in excess volume, were collected from cats that were euthanized with suspected FIP or due to other systemic diseases or very rarely on behavioural grounds. Some samples were included in earlier studies [20,21]. Collection of cerebrospinal fluid (CSF), where possible, was performed immediately following euthanasia as part of a clinical training programme and was independent of presence of presenting neurological signs, which were not specifically evaluated.…”

Discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis

“…Molecular methods have been used for detection of FCoV RNA in the effusions of cats with suspected FIP (Gut et al, 1999;Simons et al, 2004;Hornyák et al, 2012;Soma et al, 2013;Doenges et al, 2017;Felten et al, 2017;Longstaff et al, 2017). However, these methods display similar issues related to the diagnostic performances (lack of sensitivity and specificity).…”

Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus

“…The FCoV is faecally-orally transmitted, worldwide distributed and highly prevalent in feline populations (Addie et al, 2009). Since most of the molecular and serological tools available up to date are not able to distinguish between the not mutated and the mutated pathogenic form of the FCoV, the use of reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis of FIP is still extensively discussed Doenges et al, 2016;Felten et al, 2015;Longstaff et al, 2015). On the other hand, PCR is an extremely useful tool for the diagnosis of FCoV infection and shedding and consequently for the identification of shedders, when performed on faeces (Pedersen, 2014).…”