Angelica Stranieri

High diagnostic accuracy of the Sysmex XT-2000iV delta total nucleated cells on effusions for feline infectious peritonitis.

Background The ΔWBC (the ratio between DIFF and BASO counts of the Sysmex XT‐2000iV), hereafter defined as ΔTNC (total nucleated cells), is high in effusions due to feline infectious peritonitis (FIP), as cells are entrapped in fibrin clots formed in the BASO reagent. Similar clots form in the Rivaltas test, a method with high diagnostic accuracy for FIP. Objectives The objective of this study was to determine the diagnostic accuracy for FIP and the optimal cutoff of ΔTNC. Methods After a retrospective search of our database, DIFF and BASO counts, and the ΔTNC from cats with and without FIP were compared to each other. Sensitivity, specificity, and positive and negative likelihood ratios (LR+, LR−) were calculated. A ROC curve was designed to determine the cutoff for best sensitivity and specificity. Results Effusions from 20 FIP and 31 non‐FIP cats were analyzed. The ΔTNC was higher (P < .001), and BASO and DIFF counts were lower (P < .001 and P < .05) in FIP than in non‐FIP cats. Only 2 FIP cats with atypical effusions had a ΔTNC < 3.0. The cutoff identified by the ROC curve (area under curve: 0.94; P < .001) was 1.7 (Sensitivity = 90.0%; Specificity = 93.53%; LR+ = 13.9; LR− = 0.1). A ΔTNC > 2.5 had 100% specificity. Conclusions The ΔTNC has a high diagnostic accuracy for FIP‐related effusions by providing an estimate of precipitable proteins, as the Rivaltas test, in addition to the cell count. As fibrin clots result in false lower BASO counts, the ΔTNC is preferable to the WBC count generated by the BASO channel alone in suspected FIP effusions.

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

Abstract The Feline coronavirus (FCoV) is the etiological agent of feline infectious peritonitis (FIP), a lethal disease of felids. The role of molecular methods is controversial for the diagnosis of FIP, while essential for the identification of the shedders. Thus, a fast and inexpensive method for the detection of FCoV could be beneficial, especially in multicat environments. A reverse transcription loop mediated isothermal amplification (RT-LAMP) assay was developed. RNA extraction and RT-nPCR for FCoV were performed on thirty-two samples (11 faeces, 9 blood, 8 effusions, and 4 lymph nodes) collected from 27 cats. Six RT-LAMP primers were designed from the same conserved region of RT-nPCR, and the assay was run at 63°C for one hour. Results were evaluated through both agarose gel run and hydroxynapthol blue (HNB) dye and then compared with RT-nPCR results for the assessment of sensitivity and specificity. The overall specificity was 100%, but the sensitivity was 50% and 54.5% for agarose gel and HNB respectively. Therefore, RT-LAMP seems optimal to confirm the presence of the virus, but not applicable to exclude it.

Frequency of electrophoretic changes consistent with feline infectious peritonitis in two different time periods (2004-2009 vs 2013-2014).

Objectives The aim of this study was to evaluate whether the frequency of electrophoretic changes in serum of cats with feline infectious peritonitis (FIP) changed in recent years vs past years. Methods Agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) from cats with FIP and healthy cats recorded in the periods 2004–2009 and 2013–2014 were retrospectively analysed. Relative and absolute values of each electrophoretic fraction were recorded and the number of cats showing single or combined electrophoretic changes consistent with FIP (hypoalbuminaemia, inverted albumin to globulin [A:G] ratio, increased total protein, total globulin, alpha [α]2-globulin and gamma [γ]-globulin concentration) were counted. Additionally, a visual analysis of electrophoretograms was also performed. Results for the two time periods were statistically compared. Results The details of 91 AGE procedures (41 from cats with FIP and 50 from healthy cats) and 45 CZE procedures (26 from cats with FIP and 19 from healthy cats) were obtained from the database. No significant differences between the two time periods were found both in FIP and in healthy cats analysed with CZE and in healthy cats analysed with AGE. Compared with 2004–2009, cats with FIP sampled in 2013–2014 with AGE showed a significantly lower concentration of total protein, γ-globulins and total globulins, and a significantly higher A:G ratio and percentage of albumin and α2-globulins. Using both AGE and CZE, in recent years the proportion of cats with high α2-globulins without gammopathy and the proportion of cats with gammopathy alone decreased. With a visual approach, the number of patterns considered as dubious increased in the second period with AGE (non-statistically significant). Conclusions and relevance The frequency of electrophoretic abnormalities in cats with FIP decreased in recent years, independently of the technique employed. Although the mechanism responsible for this change was not investigated in this study, this altered frequency may decrease the diagnostic accuracy of serum protein electrophoresis for FIP.

Sysmex XT-2000iV scattergram analysis in a cat with basophilia

A 13-year-old female Domestic Shorthair cat was presented to the Veterinary Teaching Hospital of the University of Milan for an interscapular mass suspected to be a mesenchymal malignant tumor. A preoperative CBC performed with Sysmex XT-2000iV showed leukocytosis with neutrophilia and eosinophilia. The Sysmex WBC/DIFF scattergram showed an additional, well-separated cluster of events between the neutrophil, eosinophil, and lymphocyte clusters. Blood smear evaluation revealed the presence of a significant number of basophils; thus, it was hypothesized that the additional cluster could represent the basophilic population. A second CBC, 24 days later, showed the same pattern on the WBC/DIFF scattergram in the absence of leukocytosis and neutrophilia. After surgical excision of the mass, a definitive diagnosis of feline injection site sarcoma was made. To the authors knowledge, there are no previous reports about the identification of feline basophils in the WBC/DIFF scattergram of Sysmex XT-2000iV.

Comparison of the performance of laboratory tests in the diagnosis of feline infectious peritonitis

We compared the performance of clinicopathologic and molecular tests used in the antemortem diagnosis of feline infectious peritonitis (FIP). From 16 FIP and 14 non-FIP cats, we evaluated retrospectively the sensitivity, specificity, and likelihood ratios (LRs) of serum protein electrophoresis, α1-acid glycoprotein (AGP) on peripheral blood, screening reverse-transcription nested PCR (RT-nPCR) on the 3’–untranslated region (3’-UTR), and spike (S) gene sequencing on peripheral blood, body cavity effusions, and tissue, as well as body cavity cytology and delta total nucleated cell count (ΔTNC). Any of these tests on blood, and especially the molecular tests, may support or confirm a clinical diagnosis of FIP. A negative result does not exclude the disease except for AGP. Cytology, 3’-UTR PCR, and ΔTNC may confirm a clinical diagnosis on effusions; cytology or 3’-UTR PCR may exclude FIP. Conversely, S gene sequencing is not recommended based on the LRs. On tissues, S gene sequencing is preferable when histology is highly consistent with FIP, and 3’-UTR PCR when FIP is unlikely. Combining one test with high LR+ with one with low LR− (e.g., molecular tests and AGP on blood, ΔTNC and cytology in effusions) may improve the diagnostic power of the most used laboratory tests.

Diagnosing feline infectious peritonitis using the Sysmex XT-2000iV based on frozen supernatants from cavitary effusions

The delta total nucleated cells (ΔTNC) measurement with the Sysmex XT-2000iV (Sysmex Europe, Norderstedt, Germany) has high diagnostic accuracy on effusions in feline infectious peritonitis (FIP) cases, but the test can be performed only on fresh samples. We evaluated whether supernatants from effusions retain the ability to induce cell clumping and assessed the diagnostic accuracy of this modified ΔTNC method. Effusions were collected from FIP cats (n = 19) and from cats with other diseases (n = 15). ΔTNC was measured on fresh samples and on frozen–thawed supernatants after the addition of feline blood at 1:10 dilution. Diagnostic accuracy was assessed at the cutoffs of suggestive of FIP (ΔTNC = 1.7) and consistent with FIP (ΔTNC = 3.4). The influence of the protein content, number of added cells, and magnitude of dilution were also investigated. Specificity and positive predictive value were 100% for both the methods. Sensitivity and negative predictive value were higher for the modified ΔTNC (84.2% and 83.3%, respectively, at the cutoff of 1.7; 78.9% and 78.9%, respectively, at the cutoff of 3.4) than for the ΔTNC on fresh samples (78.6% and 81.3%, respectively, at the cutoff of 1.7; 57.1% and 68.4%, respectively, at the cutoff of 3.4). Protein content, total cell count of the added blood, and magnitude of dilutions did not influence the results. Supernatants of frozen effusions from FIP cats retain the ability to induce cell clumping, thus the modified ΔTNC measurement is a reliable tool to diagnose FIP on samples that cannot be analyzed immediately.

A reverse transcription loop-mediated isothermal amplification (LAMP) assay for the detection of feline Coronavirus

Loop-mediated isothermal amplification (LAMP) is a molecular method that amplifies DNA under isothermal conditions. It relies on the use of 4 different primers recognizing 6 regions of the template sequence and on the use of a DNA polymerase with strand displacement activity (Notomi et al., 2000). The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002). The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensive detection of feline Coronavirus (FCoV). Six primers binding the conserved 3’UTR region of the FCoV were designed with the Primer Explorer software. Thirty-two samples of RNA (11 feces, 8 effusions, 9 blood samples and 4 tissues) on which a reverse transcription polymerase chain reaction (RT-PCR) for the 3’UTR region was performed were used. The reaction was carried out in 25μL reaction volume and the mixture was incubated in a thermocycler at 63°C for 1 hour followed by 10 minutes at 80°C. LAMP products were visualized under UV after electrophoresis migration on a 1.5% agarose gel stained with ethidium bromide, where they produce a ladder-like pattern if positive. Results where compared with those obtained on standard PCR. Sensitivity and specificity were respectively 60% and 100% on feces, 40% and 100% on effusions, 25% and 100% on blood, and 100% and 100% on tissues. The overall sensitivity and specificity of this method were of 57.1% and 100%, thus limiting a clinical application of this method, except for tissues.

Comparison between the diagnostic accuracy of clinico-pathological and molecular tests for feline infectious peritonitis (FIP)

The aim of this study was to compare the diagnostic accuracy for feline infectious peritonitis (FIP) of conventional clinic-pathological tests with that of molecular tests such as routine PCR and PCR followed by the sequencing of the Spike (S) gene. Blood, effusion and tissues specimens were collected from 21 FIP suspected cats. In vivo examination consisted of CBC, serum protein electrophoresis, AGP measurement, cytological and biochemical examination and the evaluation of the ΔTNC on effusions, and of molecular tests such the screening PCR (target: 3’UTR region) and the PCR directed towards the S gene followed by the amplification products sequencing in order to detect the aminoacidic substitution recently considered diagnostic for FIP 1 . These molecular techniques were applied to tissues collected during necropsy, which also allowed forming an FIP group (13 cats) and a non-FIP group (5 cats) based on histology and immunohistochemistry. The best test on tissues was immunohistochemistry (sens: 92.3%; spec: 100%), while the screening PCR suffered of low specificity (spec: 33.3%) and the S gene sequencing showed low sensitivity (sens: 69.2%).On effusions, the best tests resulted screening PCR and cytology (sens and spec: 100%) in comparison with the ΔTNC measurement (sens: 85.7 %; spec: 100%) and the S gene sequencing (sens: 42.8%; spec: 100%).On blood, the best test resulted AGP measurement (sens: 81.8%; spec: 100%), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: 41.7%). Screening PCR (sens: 55.6%; spec: 100%) and S gene sequencing (sens: 33.3%; spec: 100%) proved again low accuracy.