Hugo Lima de Carvalho

Feline infectious peritonitis (FIP) can be overlooked in older cats with competing causes of effusion. An older female domestic shorthair cat with hyperthyroidism and a hypertrophic cardiomyopathy phenotype developed abdominal effusion in December 2023. Initial effusion total protein was 5.46 g/dL, the albumin-to-globulin ratio was 0.48 and delta total nucleated cell ratio (ΔTNC) was 60.58, yielding a retrospectively calculated FIP Effusion Index of 126.2. Coronavirus RT-PCR detected FCoV RNA, with M1058L mutation positivity, and cell tube block immunohistochemistry (CTB-IHC) demonstrated viral antigen within macrophages. A second sample collected from scant residual effusion 9 days after the initiation of GS-441524 had a ΔTNC of 2.15 and a FIP Effusion Index of 7.17, despite a higher total protein concentration and a lower albumin-to-globulin ratio. GS-441524 treatment began on 20 December 2023 and ended on 22 March 2024. Abdominal fluid was no longer detected at the assessment on 25 January. No confirmed FIP relapse was documented before death during cardiopulmonary decompensation in January 2026. This case illustrates complementary contributions of the FIP Effusion Index and CTB-IHC, with an exploratory observation of serial FIP Effusion Index change. Threshold comparison is retrospective. The lower index was observed during treatment alongside markedly reduced effusion, but this observation does not establish a treatment effect or validate treatment monitoring.

Feline infectious peritonitis (FIP) remains diagnostically challenging, and rapid antemortem confirmation is increasingly important in the era of effective antiviral therapy. This pilot diagnostic accuracy study assessed agreement between effusion RT-qPCR and cell tube block immunohistochemistry (CTB-IHC) in 40 cats with suspected FIP and developed an exploratory effusion-based predictive score using CTB-IHC as the reference standard. RT-qPCR and CTB-IHC showed almost perfect agreement (κ = 0.86): 30 cats were positive by both methods, eight were negative by both, and two were CTB-IHC-positive/RT-qPCR-negative. RT-qPCR showed 93.8% sensitivity (95% CI: 79.2–99.2%), 100% specificity (63.1–100%), and 95.0% accuracy (83.1–99.4%). The three-parameter FIP Predictive Score, combining globulin concentration, FIP Effusion Index, and A:G ratio, achieved an apparent AUC of 0.916 (95% CI: 0.842–0.990), with 78.1% sensitivity (60.0–90.7%) and 100% specificity (63.1–100%) at ≥ 2 points. Given that the score was derived and evaluated within the same small cohort, independent external validation is essential before clinical implementation. Nevertheless, neither a low score nor a negative molecular result should independently close investigation when FIP remains clinically suspected. These findings provide a foundation for a structured, multimodal effusion-based diagnostic approach integrating molecular, antigen, biochemical, and cytometric evidence.

Feline infectious peritonitis (FIP) can be overlooked in older cats with competing causes of effusion. An older female domestic shorthair cat with hyperthyroidism and a hypertrophic cardiomyopathy phenotype developed abdominal effusion in December 2023. Initial effusion total protein was 5.46 g/dL, the albumin-to-globulin ratio was 0.48 and delta total nucleated cell count ratio (ΔTNC) was 60.58, yielding an FIP Effusion Index of 126.2. Coronavirus RT-PCR detected FCoV RNA, with M1058L mutation positivity, and cell tube block immunohistochemistry (CTB-IHC) demonstrated viral antigen within macrophages. A second effusion sample collected 9 days after the first had ΔTNC of 2.15 and a FIP Effusion Index of 7.17, despite higher total protein and a lower albumin-to-globulin ratio. GS-441524 treatment began on 20 December 2023 and ended on 22 March 2024. Abdominal fluid was no longer detected at the assessment on 25 January. No confirmed FIP relapse was documented before death during cardiopulmonary decompensation in January 2026. This case illustrates complementary contributions of the FIP Effusion Index and CTB-IHC, with an exploratory observation of serial FIP Effusion Index change. Threshold comparison is retrospective, and the serial measurements do not establish a validated marker of treatment response.

Serum protein capillary electrophoresis can characterise dysproteinaemia in dogs tested for canine leishmaniosis (CanL) as an integrated clinicopathological phenotype of humoral immune activation. This retrospective laboratory-based study evaluated serum protein electrophoretic patterns in 5544 dogs tested for CanL in Portugal between 2009 and 2026. Anti-Leishmania antibody results were classified into five ordered categories: negative, inconclusive, low positive, positive/high positive and very high positive. Total protein, albumin, alpha-1, alpha-2, beta and gamma fractions, albumin-to-globulin and albumin-to-gamma-globulin ratios were analysed. A separately defined reference subpopulation of 254 clinically healthy seronegative dogs aged ≥ 1 to p < 0.001), with lower albumin-to-globulin (0.7 vs. 1.0; p < 0.001) and albumin-to-gamma-globulin ratios (1.9 vs. 4.2; p < 0.001). Across the ordinal serological scale, gamma globulins increased from 0.8 to 2.1 g/dL (p < 0.001), whereas the albumin-to-gamma-globulin ratio decreased from 4.2 to 1.2. Gamma globulins showed the strongest ordinal association (ρ = 0.522; p < 0.001) and moderate single-marker discrimination of seropositivity (area under the receiver operating characteristic curve = 0.768; 95% confidence interval: 0.755–0.780). These findings define the CanL proteinogram as a progressive, quantifiable immunopathological phenotype, dominated by gamma-region expansion and reciprocal albumin decline, supporting serum protein electrophoresis as a reference tool for interpretation and monitoring.

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.

Canine distemper virus (CDV), currently classified within the species Morbillivirus canis, is a major vaccine-preventable pathogen of domestic dogs and a wide range of susceptible wildlife species. Still, laboratory-confirmed epidemiological data from Portugal remain scarce. This retrospective study investigated CDV molecular detection in 637 diagnostic samples from dogs with clinical suspicion of canine distemper, received from 190 veterinary medical centres across Portugal between 2013 and 2025. Cerebrospinal fluid, EDTA-anticoagulated whole blood, rectal swabs, and conjunctival swabs were tested for CDV RNA using a reverse transcription real-time PCR (RT-qPCR) assay in a qualitative approach. Overall, 215 submissions were CDV RT-qPCR-positive, corresponding to a positivity of 33.8% (95% confidence interval: 30.2–37.5%). Positivity was not significantly associated with sex, age, or Nomenclature of Territorial Units for Statistics level 2 (NUTS 2) region, but differed significantly according to specimen type, with the highest detection observed in EDTA-anticoagulated whole blood and conjunctival swabs. Mixed-breed dogs were over-represented among submitted samples and positive cases, probably reflecting management, exposure, and vaccination-related factors rather than intrinsic breed susceptibility. The central finding was a pronounced post-2020 decline in CDV RT-qPCR positivity, with very low or absent annual detection between 2021 and 2025. This pattern indicates reduced molecular detection within a passive diagnostic population but should not be interpreted as evidence of national elimination. Continued vaccination and strengthened surveillance at the domestic dog–wildlife interface remain essential.

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.

Bartonella henselae is a flea-borne zoonotic bacterium for which domestic cats constitute the principal reservoir. However, contemporary molecular epidemiological data from Portugal remain scarce. This retrospective laboratory study analysed EDTA-stabilised blood samples from apparently healthy cats submitted for routine screening by 74 veterinary centres across mainland Portugal and autonomous regions over an 11-year period (2015–2025). DNA extracts were tested using a species-specific TaqMan qPCR assay for B. henselae with an internal extraction control, and a subset of samples was subsequently confirmed by nested PCR followed by Sanger sequencing (ribC). Among 270 cats, 47 tested positive, yielding a qPCR prevalence of 17.4% (95% confidence interval [CI] 13.1–22.5). Submissions were predominantly from Northern Portugal, and infection status was not statistically associated with the Nomenclature of Territorial Units for Statistics (NUTS) level 2 region (p = 0.478). Infection was more frequent in younger cats (median age 2 years, interquartile range [IQR] 1–5; p = 0.037), while sex (p = 0.103) and breed (p = 0.730) were not significantly associated with infection status. These findings support endemic circulation of B. henselae in Portuguese cats at levels comparable to other temperate European regions. The detection of subclinical infection in apparently healthy cats is relevant to transfusion medicine and supports the inclusion of B. henselae qPCR screening in donor selection protocols.

Brucella canis is a neglected zoonotic pathogen associated with canine reproductive disorders and emerging public health concerns. This study presents the first nationwide systematic review and meta-analysis of Brucella spp. in Portugal, integrated with a 13-year retrospective seroepidemiological investigation (2013–2025) of B. canis in dogs across mainland Portugal and Insular Autonomous Regions. Among 132 canine serum samples, a seropositivity of 23.48% was observed using an immunochromatographic assay confirmed by indirect immunofluorescence (IFAT). Significant associations were identified with seasonality (p < 0.001) and breed (p = 0.001), while sex and age were not statistically significant. Municipal-level analysis revealed marked heterogeneity, with Trofa showing the highest seropositivity (58.82%) and a pooled odds ratio of 11.28 (95% CI: 2.90–43.94; p < 0.001). In parallel, meta-analyses of published data estimated a pooled seroprevalence of 2.42% in animals (95% CI: 1.79–3.13) and 10.57% in humans (95% CI: 8.80–12.47), underscoring the broader burden of Brucella spp. exposure in Portugal. These findings suggest underdiagnosis of canine brucellosis and highlight the need for enhanced surveillance in high-risk breeds and regions. The study reinforces the importance of integrated One Health strategies to improve early detection, control, and prevention of B. canis infection in both veterinary and public health contexts.

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.