Immunolabeling on Romanowsky-stained cytology (RSC) slides can be used, although there is limited evidence of its suitability for phenotyping canine and feline lymphomas. A comparison with matched cell blocks (CB) is missing. Immunolabeling on RSC and CB was compared for lymphoid markers (CD3 and PAX5) in 53 lymphomas and 4 chylous effusions from dogs and cats. The influence of pre-analytical variables (species, time of archive, type of specimens and coverslipping) and the interobserver agreement among the 2 observers was assessed. Fewer CD3+ lymphocytes were identified in RSC, while the PAX5 positivity by RSC and CB had a substantial agreement. Immunodetection of CD3 and the diagnosis of a T-cell population on RSC was more difficult. Lower intensity and higher background were noted in RSC. Immunophenotyping was inconclusive in 54% RSC and 19% CB. The interobserver reproducibility of immunophenotyping on CB was substantial, being higher than in RSC. The immunolabeling performance on the RSC of effusion and feline samples was unsatisfactory. The detection of lymphoid markers, especially membranous antigens in retrospective RSC, is affected by the pre-analytical variables: species, time of the archive, and type of specimens. CB are a more consistent type of sample for immunophenotyping purposes.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led the medical and scientific community to explore the pathogenesis and clinical manifestations of coronaviruses. In felines, a widespread coronavirus known as feline coronavirus (FCoV) can lead to feline infectious peritonitis (FIP), a highly fatal disease characterised by severe systemic inflammation. Diagnosing FCoV remains challenging due to the limited accuracy of the available methods. The present study introduces the FIP Effusion Index, a novel diagnostic method that combines the albumin-to-globulin (ALB/GLOB) ratio with the delta total nucleated cell (∆TNC) count obtained via flow cytometry using the Sysmex XN-1000V^® analyser in effusions. Samples from cats (n = 50) with suspected FIP were analysed for ∆TNC, with findings showing that a ∆TNC ≥ 2.1 is highly indicative of FIP and a ∆TNC ≥ 4.9 can be considered diagnostic. The FIP Effusion Index enhanced diagnostic precision in our group of samples, achieving 96.3% sensitivity and 95.7% specificity for values ≥ 5.06, and reaching perfect specificity (100%) with 96.3% sensitivity for values ≥ 7.54. This combined approach surpasses the accuracy of individual parameters, establishing the FIP Effusion Index as a superior diagnostic tool for FIP, with potential applications in both veterinary and human medicine for related coronavirus diseases.

Leishmania infantum is the causative agent of canine leishmaniosis (CanL), a zoonotic disease of considerable clinical and epidemiological concern. Quantification of parasite load is valuable for clinical management, particularly in low-parasite-load cases. This pilot study evaluated the correlation between cytological and molecular techniques in bone marrow samples from dogs clinically affected with leishmaniosis. Amastigotes were quantified by two independent observers using light microscopy, and the same samples were analysed by real-time polymerase chain reaction (qPCR) employing Leishmania spp. specific commercial primers. Inter-observer agreement was moderate according to Cohen’s kappa (κ = 0.47) and substantial according to the intraclass correlation coefficient (ICC = 0.63), respectively, confirming measurement reproducibility. A very strong inverse correlation was found between parasite counts and qPCR cycle threshold (Ct) values (Spearman’s ρ = −0.89; p < 0.001). Furthermore, a robust logarithmic relationship (amastigotes/μL = 10^{(−0.158 × Ct + 7.61)}; R² = 0.99994) was established allowing direct estimation of in vivo parasite concentration from molecular data. These preliminary findings suggest that qPCR can serve as a reliable, semi-quantitative tool, offering higher sensitivity in subclinical infections. The integration of molecular quantification with cytological methods enhances diagnostic accuracy and supports personalised therapeutic decision-making, advancing clinical management of CanL in a One Health context.

The antemortem clinical and clinicopathological diagnosis of feline infectious peritonitis (FIP) remains challenging, particularly when effusions deviate from the classically protein-rich, low-to-moderate-cellularity phenotype. The FIP Effusion Index, defined as the ratio of delta total nucleated cells (ΔTNC) to the albumin-to-globulin (A/G) ratio, was developed and initially evaluated in 50 effusions. Subsequent European Advisory Board on Cat Diseases (ABCD) guidelines recommended evaluation in larger cohorts and in cats with confirmed FIP. This study retrospectively evaluated the prespecified threshold (≥7.54) in 176 independent feline effusion samples analysed on the Sysmex XN-1000V and classified by effusion FCoV RT-qPCR, without independent confirmation of FIP status. Relative to this molecular reference, the FIP Effusion Index yielded a sensitivity of 97.8% (95% confidence interval [CI], 92.4–99.7%), a specificity of 88.1% (95% CI, 79.2–94.1%) and an area under the receiver operating characteristic curve (AUC) of 0.962 (95% CI, 0.928–0.995). Discrimination exceeded that of ΔTNC alone (AUC, 0.947; ΔAUC, 0.014; p = 0.012) and A/G alone (AUC, 0.887; ΔAUC, 0.074; p = 0.012). In exudates, sensitivity remained 100% (95% CI, 80.5–100%) and specificity was 82.8% (95% CI, 64.2–94.2%). Among reference-negative effusions, false-positive classification was more frequent in septic than in non-septic samples (6/19 vs. 4/65; odds ratio, 7.04; 95% CI, 1.74–28.53; p < 0.01). These findings demonstrate that the prespecified FIP Effusion Index threshold retained very high sensitivity and strong discrimination for molecular-reference positivity across a broader spectrum of feline effusions. Future studies should evaluate the FIP Effusion Index within a sequential in vivo diagnostic algorithm, using negative results to support rule-out and positive results to prompt confirmation by FCoV antigen detection using immunocytochemistry and/or immunohistochemistry.

In 2022, the World Health Organization (WHO) published the Fungal Priority Pathogens List (FPPL), yet its application has remained largely focused on human medicine, with limited consideration of animal hosts and veterinary diagnostics. This retrospective study aimed to characterise the occurrence of WHO critical- and high-priority yeasts in veterinary clinical submissions in Portugal within an explicit One Health framework. All yeast-positive submissions received by a Portuguese veterinary diagnostic laboratory between 2019 and 2026 were reviewed. Isolates were identified phenotypically, by an automated identification system and by MALDI-TOF mass spectrometry. Data on host species, sample type and year of submission were analysed using standard descriptive and inferential statistics. Among 2033 mycological submissions, 219 were yeast-positive. Out of these, 82 isolates (37.4%) corresponded to WHO critical- or high-priority taxa, most frequently Candida albicans, followed by Nakaseomyces glabratus, Cryptococcus neoformans var. neoformans, the Candida parapsilosis complex and Candida tropicalis. The remaining 137 isolates (62.6%) corresponded to non-WHO taxa, among which the most frequent were Papiliotrema laurentii and Debaryomyces hansenii (n = 21 each; 15.3%), followed by the Stephanoascus ciferrii complex (n = 15; 11.0%), Meyerozyma guilliermondii and Candida sake (n = 12 each; 8.8%), and Wickerhamomyces anomalus (n = 9; 6.6%). WHO-prioritised taxa were recovered predominantly from ornamental birds, as well as from dogs, cats and marine mammals. These findings demonstrate that FPPL-listed yeasts are regularly detected among yeast-positive veterinary diagnostic submissions and highlight ornamental birds as prominent hosts within this dataset.