Safety evaluation of the food enzyme sucrose phosphorylase from the genetically modified Escherichia coli strain LE1B109-pPB129.

The food enzyme sucrose phosphorylase (sucrose: phosphate α- d-glucosyltransferase; EC 2.4.1.7) is produced with the genetically modified Escherichia coli strain LE1B109-pPB129 by c-LEcta GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was free from viable cells of the production organism. It is intended to be used in combination with a cellobiose phosphorylase in the production of the specialty carbohydrate cellobiose. Since residual amounts of food enzyme-total organic solids are removed by the downstream purification steps, the Panel considered that toxicological studies other than assessment of allergenicity were unnecessary and a dietary exposure was not estimated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Steroidal epoxides as anticancer agents in lung, prostate and breast cancers: The case of 1,2-epoxysteroids.

Cancer continues to be a serious threat to human health worldwide. Lung, prostate and triple-negative breast cancers are amongst the most incident and deadliest cancers. Steroidal compounds are one of the most diversified therapeutic classes of compounds and they were proven to be efficient against several types of cancer. The epoxide function has been frequently associated with anticancer activity, particularly the 1,2-epoxide function. For this reason, three 1,2-epoxysteroid derivatives previously synthesised (EP1, EP2 and EP3) and one synthesised for the first time (oxysteride) were evaluated against H1299 (lung), PC3 (prostate) and HCC1806 (triple-negative breast) cancer cell lines. A human non-tumour cell line, MRC-5 (normal lung cell line) was also used. EP2 was the most active compound in all cell lines with IC50 values of 2.50, 3.67 and 1.95 µM, followed by EP3 with IC50 values of 12.65, 15.10 and 14.16 µM in H1299, PC3 and HCC1806 cells, respectively. Additional studies demonstrated that EP2 and EP3 induced cell death by apoptosis at lower doses and apoptosis/necrosis at higher doses, proving that their effects were dose-dependent. Both compounds also exerted their cytotoxicity by ROS production and by inducing double-strand breaks. Furthermore, EP2 and EP3 proved to be much less toxic against a normal lung cell line, MRC5, indicating that both compounds might be selective, and they also demonstrated suitable in silico ADME and toxicity parameters. Finally, none of the compounds induced haemoglobin release. Altogether, these results point out the extreme relevance of both compounds, especially EP2, in the potential treatment of these types of cancer.

Corrigendum: Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

[This corrects the article DOI: 10.3389/fmolb.2023.1082915.].

Anti-Obesity Therapeutic Targets Studied In Silico and In Vivo: A Systematic Review.

In the age of information technology and the additional computational search tools and software available, this systematic review aimed to identify potential therapeutic targets for obesity, evaluated in silico and subsequently validated in vivo. The systematic review was initially guided by the research question "What therapeutic targets have been used in in silico analysis for the treatment of obesity?" and structured based on the acronym PECo (P, problem; E, exposure; Co, context). The systematic review protocol was formulated and registered in PROSPERO (CRD42022353808) in accordance with the Preferred Reporting Items Checklist for Systematic Review and Meta-Analysis Protocols (PRISMA-P), and the PRISMA was followed for the systematic review. The studies were selected according to the eligibility criteria, aligned with PECo, in the following databases: PubMed, ScienceDirect, Scopus, Web of Science, BVS, and EMBASE. The search strategy yielded 1142 articles, from which, based on the evaluation criteria, 12 were included in the systematic review. Only seven these articles allowed the identification of both in silico and in vivo reassessed therapeutic targets. Among these targets, five were exclusively experimental, one was exclusively theoretical, and one of the targets presented an experimental portion and a portion obtained by modeling. The predominant methodology used was molecular docking and the most studied target was Human Pancreatic Lipase (HPL) (n = 4). The lack of methodological details resulted in more than 50% of the papers being categorized with an "unclear risk of bias" across eight out of the eleven evaluated criteria. From the current systematic review, it seems evident that integrating in silico methodologies into studies of potential drug targets for the exploration of new therapeutic agents provides an important tool, given the ongoing challenges in controlling obesity.

Safety evaluation of the food enzyme cellobiose phosphorylase from the genetically modified Escherichia coli strain LE1B109-pPB130.

The food enzyme cellobiose phosphorylase (cellobiose: phosphate α-d-glucosyltransferase; EC 2.4.1.20) is produced with the genetically modified Escherichia coli strain LE1B109-pPB130 by c-LEcta GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in combination with a sucrose phosphorylase in the production of the specialty carbohydrate cellobiose. Since residual amounts of total organic solids are removed by downstream purification steps, the Panel considered that toxicological studies other than assessment of allergenicity were unnecessary and a dietary exposure was not estimated. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The association between gender equality and climate adaptation across the globe.

INTRODUCTION: Climate change has a disproportionate impact on women in comparison to men, and women have a key role to play in climate adaptation. However, evidence is lacking on how gender inequalities may be associated with climate vulnerability and ability to respond at country level. METHODS: This ecological study investigated the association between climate adaptation, measured by the Notre Dame Global Adaptation Initiative Country Index (ND-GAIN), and gender equality, measured by the Global Gender Gap Index (GGGI) developed by the World Economic Forum and the Gender Inequality Index (GII) developed by the United Nations. Simple linear regression was used to estimate the associations between the indices and their subdomains for 146 countries. RESULTS: There was an approximately linear association between the GGGI and climate adaptation. Each 1% increase in gender equality was associated with a 0.6% increase in the ND-GAIN score (the slope was 0.59, with a 95% confidence interval [0.33 to 0.84]). This was driven by a negative association between gender equality and vulnerability (-0.41 [-0.62 to -0.20]), and a positive association between gender equality and readiness (0.77 [0.44 to 1.10]). The strongest associations between gender equality and climate adaptation were observed for the education domain of the GGGI. There was a strong negative linear association between the GII and climate adaptation, which explained most (86%) of the between-country variation in climate adaptation. Each 1% increase in gender inequality was associated with a 0.5% decrease in the ND-GAIN score (-0.54 [-0.57 to -0.50]). The association between gender inequality and readiness was stronger than the association with vulnerability (0.41 [0.37 to 0.44] for vulnerability versus - 0.67 [-0.72 to -0.61] for readiness). CONCLUSIONS: Gender inequality, measured broadly across different domains of life, is associated with climate adaptation at country level, both in terms of vulnerability to impact and readiness to respond.

Siderophores and metallophores: Metal complexation weapons to fight environmental pollution.

Siderophores are small molecules of organic nature, released by bacteria to chelate iron from the surrounding environment and subsequently incorporate it into the cytoplasm. In addition to iron, these secondary metabolites can complex with a wide variety of metals, which is why they are commonly studied in the environment. Heavy metals can be very toxic when present in large amounts on the planet, affecting public health and all living organisms. The pollution caused by these toxic metals is increasing, and therefore it is urgent to find practical, sustainable, and economical solutions for remediation. One of the strategies is siderophore-assisted bioremediation, an innovative and advantageous alternative for various environmental applications. This research highlights the various uses of siderophores and metallophores in the environment, underscoring their significance to ecosystems. The study delves into the utilization of siderophores and metallophores in both marine and terrestrial settings (e.g. bioremediation, biocontrol of pathogens, and plant growth promotion), such as bioremediation, biocontrol of pathogens, and plant growth promotion, providing context for the different instances outlined in the existing literature and highlighting their relevance in each field. The study delves into the structures and types of siderophores focusing on their singular characteristics for each application and methodologies used. Focusing on recent developments over the last two decades, the opportunities and challenges associated with siderophores and metallophores applications in the environment were mapped to arm researchers in the fight against environmental pollution.

CD5L as a promising biological therapeutic for treating sepsis.

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L's important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L-/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans.

Wastewater Disinfection with Photodynamic Treatment and Evaluation of its Ecotoxicological Effects.

Research has demonstrated the presence of viruses in wastewater (WW), which can remain viable for a long period, posing potential health risks. Conventional WW treatment methods involving UV light, chlorine and ozone efficiently reduce microbial concentrations, however, they produce hazardous byproducts and microbial resistance that are detrimental to human health and the ecosystem. Hence, there is a need for novel disinfection techniques. Antimicrobial Photodynamic Inactivation (PDI) emerges as a promising strategy, utilizing photosensitizers (PS), light, and dioxygen to inactivate viruses. This study aims to assess the efficacy of PDI by testing Methylene Blue (MB) and the cationic porphyrin TMPyP as PSs, along a low energy consuming white light source (LED) at an irradiance of 50 mW/cm2, for the inactivation of bacteriophage Phi6. Phi6 serves as an enveloped RNA-viruses surrogate model in WW. PDI experiments were conducted in a buffer solution (PBS) and real WW matrices (filtered and non-filtered). Considering the environmental release of the treated effluents, this research also evaluated the ecotoxicity of the resulting solution (post-PDI treatment effluent) on the model organism Daphnia magna, following the OECD immobilization technical 202 guideline. Daphnids were exposed to WW containing the tested PS at different concentrations and dilutions (accounting for the dilution factor during WW release into receiving waters) over 48 h. The results indicate that PDI with MB efficiently inactivated the model virus in the different aqueous matrices, achieving reductions superior to 8 log10 PFU/mL, after treatments of 5 min in PBS and of ca. 90 min in WW. Daphnids survival increased when subjected to the PDI-treated WW with MB, considering the dilution factor. Overall, the effectiveness of PDI in eliminating viruses in WW, the fading of the toxic effects on daphnids after MB' irradiation and the rapid dilution effect upon WW release in the environment highlight the possibility of using MB in WW PDI-disinfection.

Taxonomic identity of the Bacillus licheniformis strains used to produce food enzymes evaluated in published EFSA opinions.

Bacillus paralicheniformis, a species known to produce the antimicrobial bacitracin, could be misidentified as Bacillus licheniformis, depending on the identification method used. For this reason, the European Commission requested EFSA to review the taxonomic identification of formerly assessed B. licheniformis production strains. Following this request, EFSA retrieved the raw data from 27 technical dossiers submitted and found that the taxonomic identification was established by 16S rRNA gene analyses for 15 strains and by whole genome sequence analysis for 12 strains. As a conclusion, only these 12 strains could be unambiguously identified as B. licheniformis.

Continuous UV-C/H2O2 and UV-C/Chlorine applied to municipal secondary effluent and nanofiltration retentate: removal of contaminants of emerging concern, ecotoxicity, and reuse potential.

As global effects of water scarcity raise concerns and environmental regulations evolve, contemporary wastewater treatment plants (WWTPs) face the challenge of effectively removing a diverse range of contaminants of emerging concern (CECs) from municipal effluents. This study focuses on the assessment of advanced oxidation processes (AOPs), specifically UV-C/H2O2 and UV-C/Chlorine, for the removal of 14 target CECs in municipal secondary effluent (MSE, spiked with 10 µg L-1 of each CEC) or in the subsequent MSE nanofiltration retentate (NFR, no spiking). Phototreatments were carried out in continuous mode operation, with a hydraulic retention time of 3.4 min, using a tube-in-tube membrane photoreactor. For both wastewater matrices, UV-C photolysis (3.3 kJ L-1) exhibited high efficacy in removing CECs susceptible to photolysis, although lower treatment performance was observed for NFR. In MSE, adding 10 mg L-1 of H2O2 or Cl2 enhanced treatment efficiency, with UV-C/H2O2 outperforming UV-C/Chlorine. Both UV-C/AOPs eliminated the chronic toxicity of MSE toward Chlorella vulgaris. In the NFR, not only was the degradation of target CECs diminished, but chronic toxicity to C. vulgaris persisted after both UV-C/AOPs, with UV-C/Chlorine increasing toxicity due to potential toxic by-products. Nanofiltration permeate (NFP) exhibited low CECs and microbial content. A single chlorine addition effectively controlled Escherichia coli regrowth for 3 days, proving NFP potential for safe reuse in crop irrigation (< 1 CFU/100 mL for E. coli; < 1 mg L-1 for free chlorine). These findings provide valuable insights into the applications and limitations of UV-C/H2O2 and UV-C/Chlorine for distinct wastewater treatment scenarios.

Factors associated with the consumption of voluntarily fortified foods with micronutrients by the Portuguese population.

This study aimed to identify the sociodemographic and health-related factors associated with the consumption of voluntarily fortified foods with micronutrients (Mn-FF) by the Portuguese population, using data from the National Food, Nutrition and Physical Activity Survey (IAN-AF 2015-2016). Food consumption, sociodemographic and other health-related factors, and physical activity data were computerised using the You Eat & Move e-platform. Foods consumed by participants and labelled foods reported as consumed were included in the database. Mn-FF were considered all foods containing added micronutrients legally authorised, and MN-FF users were those who consumed at least one Mn-FF on at least one of the dietary recall days. Approximately 57% of the population consumed at least one Mn-FF. Children and adolescents consumed significantly more Mn-FF than older age groups. The primary contributors to Mn-FF consumption were breakfast cereals and fat spreads. Being a child (female OR 2.07 [95% CI: 1.52, 2.83]; male OR 4.80 [95% CI: 3.23, 7.14]) or adolescent (female OR 1.62 [95% CI: 1.78, 2.22]; male OR 4.59 [95% CI: 3.26, 6.47]), having a higher level of education (female OR 1.52 [95% CI: 1.17, 1.99]; male OR 1.85 [95% CI: 1.42, 2.42]) and engaging in regular physical activity (female OR 1.31 [95% CI: 1.09, 1.58]; male OR 1.36 [95% CI: 1.11, 1.68]) were factors positively associated with Mn-FF consumption. Conversely, obesity (female OR 0.76 [95% CI: 0.60, 0.96]), living in predominantly rural areas (male OR 0.70 [95% CI: 0.49, 0.91]) and eating fewer mid-meals per day (female OR 0.60 [95% CI: 0.48, 0.76]; male OR 0.64 [95% CI: 0.49, 0.84]) were associated with lower Mn-FF consumption. Younger age, higher education and physical activity practice were positively associated with Mn-FF consumption. These findings provide important insights on priority factors to be considered when deciding on nutrient fortification policies from both public health and food industry perspectives.

Predictability of cortico-cortical connections in the mammalian brain.

Despite a five order of magnitude range in size, the brains of mammals share many anatomical and functional characteristics that translate into cortical network commonalities. Here we develop a machine learning framework to quantify the degree of predictability of the weighted interareal cortical matrix. Partial network connectivity data were obtained with retrograde tract-tracing experiments generated with a consistent methodology, supplemented by projection length measurements in a nonhuman primate (macaque) and a rodent (mouse). We show that there is a significant level of predictability embedded in the interareal cortical networks of both species. At the binary level, links are predictable with an area under the ROC curve of at least 0.8 for the macaque. Weighted medium and strong links are predictable with an 85%-90% accuracy (mouse) and 70%-80% (macaque), whereas weak links are not predictable in either species. These observations reinforce earlier observations that the formation and evolution of the cortical network at the mesoscale is, to a large extent, rule based. Using the methodology presented here, we performed imputations on all area pairs, generating samples for the complete interareal network in both species. These are necessary for comparative studies of the connectome with minimal bias, both within and across species.

Evaluating the User Experience of a Smartphone-Delivered Sexual Health Promotion Program for Older Adults in the Netherlands: Single-Arm Pilot Study.

BACKGROUND: Sexual health is an important component of quality of life in older adults. However, older adults often face barriers to attaining a fulfilling sexual life because of issues such as stigma, lack of information, or difficult access to adequate support. OBJECTIVE: We aimed to evaluate the user experience of a self-guided, smartphone-delivered program to promote sexual health among older adults. METHODS: The mobile app was made available to community-dwelling older adults in the Netherlands, who freely used the app for 8 weeks. User experience and its respective components were assessed using self-developed questionnaires, the System Usability Scale, and semistructured interviews. Quantitative and qualitative data were descriptively and thematically analyzed, respectively. RESULTS: In total, 15 participants (mean age 71.7, SD 9.5 years) completed the trial. Participants showed a neutral to positive stance regarding the mobile app's usefulness and ease of use. Usability was assessed as "Ok/Fair." The participants felt confident about using the mobile app. To increase user experience, participants offered suggestions to improve content and interaction, including access to specialized sexual health services. CONCLUSIONS: The sexual health promotion program delivered through a smartphone in a self-guided mode was usable. Participants' perception is that improvements to user experience, namely in content and interaction, as well as connection to external services, will likely improve usefulness and acceptance.

Safety evaluation of the food enzyme glutaminase from the genetically modified Bacillus licheniformis strain NZYM-JQ.

The food enzyme glutaminase (l-glutamine amidohydrolase EC 3.5.1.2) is produced with the genetically modified Bacillus licheniformis strain NZYM-JQ by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The production strain met the requirements for the qualified presumption of safety (QPS). The food enzyme is free from viable cells of the production organism and its DNA. The enzyme under assessment is intended to be used in six food manufacturing processes. Dietary exposure was estimated to be up to 0.148 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concern resulting from the food enzyme manufacturing process, toxicological studies were not considered necessary. A search was made for the similarity of the amino acid sequence to those of known allergens and one match with a pollen allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, particularly for individuals sensitised to birch and oak pollen. The Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

Unveiling the consequences of early human saliva contamination on membranes for guided bone regeneration.

AIMS: GBR membranes have various surface properties designed to elicit positive responses in regenerative clinical procedures; dental clinicians attempt to employ techniques to prevent the direct interaction of contaminated oral fluids with these biomaterials. However, saliva is uninterruptedly exhibited in oral surgical procedures applying GBR membranes, suggesting a persistent interaction with biomaterials and the surrounding oral tissues. This fundamental study aimed to investigate potential alterations in the physical, chemical, and key biological properties of membranes for guided bone regeneration (GBR) caused by isolated early interaction with human saliva. METHODS: A reproducible step-by-step protocol for collecting and interacting human saliva with membranes was developed. Subsequently, membranes were evaluated for their physicochemical properties, protein quantification, DNA, and 16S rRNA levels viability of two different cell lines at 1 and 7 days, and ALP activity. Non-interacted membranes and pure saliva of donors were applied as controls. RESULTS: Qualitative morphological alterations were noticed; DNA extraction and 16S quantification revealed significantly higher values. Furthermore, the viability of HGF-1 and MC3T3-E1 cells was significantly (p < .05) reduced following saliva interaction with biodegradable membranes. Saliva contamination did not prejudice PTFE membranes significantly in any biological assay. CONCLUSIONS: These outcomes demonstrated a susceptible response of biodegradable membranes to isolated early human saliva interaction, suggesting impairment of structural morphology, reduced viability to HGF-1 and MC3T3-E1, and higher absorption/adherence of DNA/16S rRNA. As a result, clinical oral procedures may need corresponding refinements.

Chronotype, Time of Day, and Children's Cognitive Performance in Remote Neuropsychological Assessment.

Research on the influence of chronotype and time of day (TOD) on cognitive performance, especially in children, is limited. We explored potential interactive effects, hypothesizing that performance differs when comparing preferred vs. non-preferred TOD. In total, 76 morning-type (MT = 37) or evening-type (ET = 39) children from the third and fourth grades (48.7% girls; M age = 8.05; SD age = 0.51), identified through the Children Chronotype Questionnaire, completed two 30-min neuropsychological assessment sessions via videoconference on the first (9:00) or last hour (16:00) of the school day. The protocol included neuropsychological tests targeting memory, language, and attention/executive domains. The results revealed an interactive effect of medium size between chronotype and TOD on a Rapid Alternating Stimulus (Naming) Task. MT and ET performed faster in asynchrony conditions (morning for ET; afternoon for MT). Additionally, ET outperformed MT in a Backward Digit Span Task, irrespective of TOD. TOD also influenced performance on an Alternating Verbal Fluency Task, with both MT and ET children performing better in the morning. These results underscore the importance of chronotype and TOD in children's cognitive performance, particularly in working memory and verbal fluency. Children assessed during non-preferred TOD exhibited better performance on some cognitive tasks, challenging the assumption that optimal times always yield superior results.

Knowledge about cervical cancer in young Portuguese women: a cross-sectional study.

Introduction: Health literacy is crucial to adherence to medical interventions in therapeutics, prevention, and diagnosis. The basis for literacy is knowledge. To accomplish the goals for the elimination of cervical cancer, one of the most prevalent and preventable cancers, we must understand the determinants of non-adherence and address them specifically to ensure patients' active participation. Aim: To determine women's knowledge regarding the manifestations of cervical cancer and its prevention. Materials and methods: We conducted a cross-sectional study in an urban population from northern Portugal. Women aged 18 to 30 years were randomly assigned to answer the Cervical Cancer Awareness Measure questionnaire, including questions of knowledge about the causes and symptoms of cervical cancer, prospecting for individual and social-related determinants. Results: The total number of participants was 270, with a mean age of 24.7 years. Knowledge about symptoms scored 5.4 ± 2.6, with a maximum of 12 points, and knowledge about the causes scored 5.7 ± 1.9, with a maximum of 11 points. The correlation between both was 0.334. High education, high socio-economic status, self-perception of one's capacity to recognize symptoms, and knowledge about the HPV vaccine were associated with better knowledge. Discussion: Portuguese women present low knowledge about cervical cancer, potentially affecting their health through exposure to risk situations and non-adherence to routine screening.

Feline leishmaniasis in an animal shelter in northeastern Brazil: Clinical aspects, coinfections, molecular detection, and serological study of a new recombinant protein.

Infection and clinical cases of leishmaniasis caused by Leishmania infantum in cats have been increasingly reported in several countries, including Brazil. In this study, we used an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) based on a recombinant antigen (rKDDR-plus) to detect anti-Leishmania antibodies in cats from an animal shelter in northeastern Brazil. We compared the results with an ELISA using L. infantum crude antigen (ELISA-CA). We also investigated the presence of Leishmania DNA in blood or ocular conjunctival samples as well as the association between Leishmania PCR positivity and serological positivity to feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and Toxoplasma gondii. Concerning serological assays, a higher positivity was detected using the ICT-rKDDR-plus (7.5%; 7/93) as compared to ELISA-rKDDR-plus (5.4%; 5/93) and ELISA-CA (4.3%; 4/93). Upon PCR testing, 52.7% (49/93) of the ocular conjunctival swabs and 48.3% (44/91) of the blood samples were positive. Together, PCR and serological testing revealed overall positivities of 73.1% (68/93) and 12.9% (12/93), respectively. Among PCR-positive samples, 45.5% (31/68) showed co-infection with FIV, 17.6% (12/68) with FeLV, and 82.3% (56/68) with T. gondii. More than half of the PCR-positive cats showed at least one clinical sign suggestive of leishmaniasis (58.8%; 40/68) and dermatological signs were the most frequent ones (45.5%; 31/68). Both tests employing the recombinant antigen rKDDR-plus (i.e., ICT-rKDDR-plus and ELISA-rKDDR-plus) detected more positive cats than the ELISA-CA but presented low overall accuracy. PCR testing using either blood or ocular conjunctival samples detected much more positive cats than serological tests.