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- Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapyPublication . Dias, Joana N. R.; Almeida, André; S. André, Ana; Aguiar, Sandra I; Bule, Pedro; Nogueira, Sara; Oliveira, Soraia S; Carrapiço, Belmira; Gil, Solange; Tavares, Luis; Aires Da Silva, FredericoAnti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identifed six amino acid diferences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived singledomain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.
- Evaluation of a biocide used in the Biological Isolation and Containment Unit of a Veterinary Teaching HospitalPublication . GERALDES, CATARINA; Verdial, Cláudia; Cunha, Eva; Almeida, Virgilio; Tavares, Luis; Oliveira, Manuela; Gil, SolangeHospital-acquired infections (HAIs) are a rising problem worldwide, and the best way of coping with them is through infection tracking and surveillance systems, combined with prevention strategies, namely efficient disinfection protocols, that employ various biocides. However, increasing reports about reductions in biocide susceptibility and the development of cross-resistance to antimicrobials emphasize the need for identifying the factors influencing biocide efficiency. In this study, 29 bacterial isolates (n = 3 E. coli, n = 2 Pseudomonas spp., n = 23 Enterococcus spp., and n = 1 Staphylococcus pseudintermedius), obtained from environmental samples collected from the Biological Isolation and Containment Unit (BICU), of the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine, University of Lisbon, were tested in order to determine their antimicrobial susceptibility to various antibiotics. Thirteen of these isolates were further selected in order to determine their antimicrobial susceptibility to Virkon™ S, with and without the presence of organic matter. Afterward, seven of these isolates were incubated in the presence of sub-lethal concentrations of this formulation and, subsequently, new susceptibility profiles were determined. Fourteen of the 29 isolates (48.3%) were classified as multidrug resistant, all previously identified as enterococci. Concerning Virkon™ S’s susceptibility, the Minimal Bactericidal Concentration (MBC) of this biocide regarding all isolates was at least eight times lower than the concentration regularly used, when no organic matter was present. However, when organic matter was added, MBC values rose up to 23 times. After exposure to sub-lethal concentrations of Virkon™ S, four enterococci presented a phenotypical change regarding antimicrobial susceptibility towards gentamicin. Virkon™ S also resulted in higher MBC values, up to 1.5 times, in the presence of low concentrations of organic matter, but no rise in these values was observed in assays without interfering substance. Virkon™ S seemed to be an efficient formulation in eliminating all bacteria isolates isolated from the BICU. However, organic matter could represent a hindrance to this ability, which emphasizes the importance of sanitization before disinfection procedures. The changes seen in antimicrobial susceptibility could be explained by a general stress-induced response promoted by the sub-lethal levels of Virkon™ S. Additionally, when no organic matter was present, a decrease in susceptibility to this biocide seemed to be non-existent.
- Diagnostic approach and grading scheme for canine allergic conjunctivitisPublication . Delgado, Esmeralda; Gomes, Érica; Gil, Solange; Lourenço, Ana MafaldaABSTRACT: Background: In humans, allergic conjunctivitis is a well described disease. In contrast, allergic conjunctivitis has not received much attention from the veterinary community so far. Canine allergic conjunctivitis (cAC) is one of the possible manifestations associated with canine atopic dermatitis (cAD), being often underdiagnosed and undertreated. Our aim is to contribute to disease characterization and clinical stagingfor cAC severity. Results: A retrospective observational study including 122 dogs that underwent a complete ophthalmological and dermatological examinations and diagnosed with allergic conjunctivitis was conducted. A total of six ophthalmic clinical signs were considered for disease characterization and clinical staging: conjunctival hyperemia, chemosis, ocular pruritus, epiphora, seromucoid to mucopurulent discharge and keratitis, classifed from 0 (absent) to 3 (severe). Scores comprised between 1–5 were considered mild, 6–10 moderate and 11–18 severe. The majority of dogs (64%) presented with moderate cAC followed by 24% of mild stages and only 12% of severe presentations. The severity of allergic conjunctivitis was not correlated to sex or age at the time of diagnosis and all presented with a bilateral form of the disease. Chemosis (84%), hyperemia (83%) and ocular pruritus (79%) was observed in 55% of the cases. Seromucoid to mucopurulent discharge (62%) and epiphora (69%) were less frequent, with keratitis being the least encountered clinical sign (15%). The degree of keratitis showed a positive correlation with both severity and chronicity of cAC (rho=0.21–0.29, p≤0.02)). Severity of cAD and cAD were not signifcantly correlated (p-value=0.4). Discussion and conclusion: The triad hyperemia, chemosis and ocular pruritus, already known in human medicine to be a reliable way of diagnosing human allergic conjunctivitis, also proved to be important in cAC Mild forms of the disease may pass unnoticed, ocular pruritus being hard to assess in canine patients.The proposed standardized diagnostic approach and novel grading scheme for cAC may be of value for both veterinary ophthalmologists and dermatologists, as well as general practitioners.
- Controlling bacteriological contamination of environmental surfaces at the biological isolation and containment unit of a veterinary teaching hospitalPublication . Verdial, C; Carneiro, Carla; Machado, Inês; Tavares, Luis; Almeida, Virgilio; Oliveira, Manuela; Gil, SolangeBackground: The Biological Isolation and Containment Unit (BICU) is a subunit of the Teaching Hospital of the Faculty of Veterinary Medicine of the University of Lisbon, Portugal, for the admission of animals with confirmed infectious diseases or under clinical suspicion and waiting for a diagnosis. As a high-risk environment for the transmission of infectious agents, it is extremely important to implement programs for the surveillance of nosocomial microorganisms in these facilities. The purpose of this study was to evaluate the level of bacterial contamination of the BICU environmental surfaces and to implement corrective actions on disinfection protocols. Swab samples were collected from selected environmental surfaces in 3 different areas of the BICU (isolation, work, and preparatory rooms) to evaluate the total aerobic bacterial load and investigate the presence of 4 nosocomial microorganisms: vancomycin-resistant Enterococcus spp., methicillin-resistant Staphylococcus aureus, 3rd-generation cephalosporin-resistant Escherichia coli, and carbapenem-resistant Pseudomonas aeruginosa. Bacterial quantification was performed by using non-selective media, while specific selective media were used for the isolation of the target microorganisms. Isolates were identified based on their macro and microscopic characteristics and their biochemical profile. Subsequently, new disinfection protocols were implemented, and their effectiveness evaluated. Results: The surfaces with the highest bacterial load in the isolation, preparatory, and worker’s rooms were the cages, hand-held sponge, and telephone, respectively. Regarding the 4 pathogens investigated, Enterococcus spp. were the most frequently isolated (11.3%), followed by E. coli (1.5%) and P. aeruginosa (1.5%). One of the P. aeruginosa isolates obtained was resistant to imipenem. In the end, new disinfection protocols were implemented, which proved to be effective in reducing bacterial counts by 99.99% in cages and the sponge, and by 90 to 99% on the telephone. Conclusions: This study allows to conclude that the cages and the human contact surfaces were the most contaminated in the isolation rooms. Nevertheless, the new disinfection strategies seemed to be effective in reducing environmental contamination, including by some potentially nosocomial agents, although more samples must be analyzed for definitive conclusions. These results may contribute to highlight the importance of infection prevention and control measures, as fundamental tools to reduce the spread of infectious agents in the hospital environment.
- Canine parvovirus : a predicting canine model for sepsisPublication . Alves, F.; Prata, S.; Nunes, Telmo; Gomes, J.; Aguiar, Sandra; Aires da Silva, Frederico; Tavares, Luis; Almeida, Virgilio; Gil, SolangeBackground: Sepsis is a severe condition associated with high prevalence and mortality rates. Parvovirus enteritis is a predisposing factor for sepsis, as it promotes intestinal bacterial translocation and severe immunosuppression. This makes dogs infected by parvovirus a suitable study population as far as sepsis is concerned. The main objective of the present study was to evaluate the differences between two sets of SIRS (Systemic Inflammatory Response Syndrome) criteria in outcome prediction: SIRS 1991 and SIRS 2001. The possibility of stratifying and classifying septic dogs was assessed using a proposed animal adapted PIRO (Predisposition, Infection, Response and Organ dysfunction) scoring system. Results: The 72 dogs enrolled in this study were scored for each of the PIRO elements, except for Infection, as all were considered to have the same infection score, and subjected to two sets of SIRS criteria, in order to measure their correlation with the outcome. Concerning SIRS criteria, it was found that the proposed alterations on SIRS 2001 (capillary refill time or mucous membrane colour alteration) were significantly associated with the outcome (OR = 4.09, p < 0.05), contrasting with the 1991 SIRS criteria (p = 0.352) that did not correlate with the outcome. No significant statistical association was found between Predisposition (p = 1), Response (p = 0.1135), Organ dysfunction (p = 0.1135), total PIRO score (p = 0.093) and outcome. To explore the possibility of using the SIRS criteria as a fast decision-making tool, a Fast-andFrugal tree (FFT) was created with a sensitivity of 92% and a specificity of 29%. Conclusion: These results suggest that increasing the SIRS criteria specificity may improve their prognostic value and their clinical usefulness. In order to improve the proposed PIRO scoring system outcome prediction ability, more specific criteria should be added, mainly inflammatory and organ dysfunction biomarkers.
- A 5-year retrospective study of canine and feline patients referred to an isolation unit for infectious diseasesPublication . Paulo, Catarina; Machado, Inês; Carvalho, Helena; Gomes, Joana; Mota, Ana Deodato; Tavares, Luis; Almeida, Virgilio; Gil, SolangeBackground: Referral of cases is becoming more and more frequent in companion animal practice. The Infectious Diseases Isolation Unit (IDIU) admits first opinion, second opinion and referred patients with a confirmed infectious disease (ID) or a clinically suspected ID that is awaiting laboratory diagnosis. The primary aims of this study were to describe the annual number and characteristics of patients referred to the IDIU and identify the most frequent IDs in referred dogs and cats. A secondary aim was to investigate possible differences in the length of the hospitalisation and the clinical outcome among referred cases and those admitted to the IDIU after first and second opinion appointments. Methods: A retrospective study was carried out on patients hospitalised at the unit over 5 years from 9th October 2013 to 31st December 2018. Results: The study population consisted of 365 dogs and 515 cats to give a total of 880 patients hospitalised at the IDIU from October 2013 to December 2018. Among the 96 referred dogs, parvovirosis (37.7%) and leptospirosis (31.1%) were the most frequent IDs. Feline upper respiratory tract infection (38.2%) and feline leukaemia virus infections (36.4%) were the main causes in the 80 referred cats. Worrying noncompliance rates of dog (51.0%) and cat (52.5%) vaccination schedules were identified. The analysis of the length of hospitalisation in the three groups of patients was not statistically different. In both animal species there were statistically significant higher clinical discharge rates on the first opinion patients' group in comparison to referred patients and the second opinion group. Conclusions: Parvovirosis and leptospirosis in dogs and upper respiratory disease and feline leukaemia virus infection in cats were the most common diagnoses for patients admitted to the IDIU, reinforcing the need for accurate vaccination. Discharge rates results pinpoint the need for timely accurate reference.