Evaluating the In Vitro Activity and Safety of Modified LfcinB Peptides as Potential Colon Anticancer Agents: Cell Line Studies and Insect-Based Toxicity Assessments.

Anticancer peptides are increasingly being considered as alternative treatments for cancer due to their potency, selectivity, and low toxicity. Previously, the peptide LfcinB (21-25)Pal showed in vitro anticancer effects against the Caco-2 colon cancer cell line (half-maximal inhibitory concentration (IC50): 86 µM). In this study, we developed modifications to the peptide sequence to increase its anticancer activity. Sequence modifications were made such as the inclusion of amino hexanoic acid (Ahx), N-terminal biotinylation, acetylation, and substitutions of Orn for Arg and/or d-Arg by l-Arg. The molecules were synthesized using manual solid-phase peptide synthesis (SPPS), and their synthetic feasibility (SAScore) ranged from 6.2 to 7.6. The chromatographic purities of the synthesized peptides were greater than 89%. We found that Ahx-RWQWRWQWR and RWQWRWQW-Orn showed activity against both Caco-2 and HT-29 cell lines and decreased IC50 values by approx. 50% in Caco-2 cells (IC50: 40 µM) when compared to the parent peptide RWQWRWQWR. Moreover, the modified peptides demonstrated lower hemolytic effects, with values <10% at 200 µg/mL. Toxicity was assessed using the Galleria mellonella model and the half-maximal lethal dose (LD50) for the best peptides was >100 mg/kg, indicating that their toxicity is classified as moderately toxic or lower. In contrast, cisplatin showed an LD50 of 13 mg/Kg. The designed anticancer peptides presented good in vitro activity and low toxicity, making them promising molecules for future drug development studies.

Cell Aggregation Capability of Clinical Isolates from Candida auris and Candida haemulonii Species Complex.

The opportunistic fungal pathogens belonging to the Candida haemulonii complex and the phylogenetically related species Candida auris are well-known for causing infections that are difficult to treat due to their multidrug-resistance profiles. Candida auris is even more worrisome due to its ability to cause outbreaks in healthcare settings. These emerging yeasts produce a wide range of virulence factors that facilitate the development of the infectious process. In recent years, the aggregative phenotype has been receiving attention, as it is mainly associated with defects in cellular division and its possible involvement in helping the fungus to escape from the host immune responses. In the present study, we initially investigated the aggregation ability of 18 clinical isolates belonging to the C. haemulonii species complex (C. haemulonii sensu stricto, C. duobushaemulonii, and C. haemulonii var. vulnera) and C. auris. Subsequently, we evaluated the effects of physicochemical factors on fungal aggregation competence. The results demonstrated that cell-to-cell aggregation was a typically time-dependent event, in which almost all studied fungal isolates of both the C. haemulonii species complex and C. auris exhibited high aggregation after 2 h of incubation at 37 °C. Interestingly, the fungal cells forming the aggregates remained viable. The aggregation of all isolates was not impacted by pH, temperature, ß-mercaptoethanol (a protein-denaturing agent), or EDTA (a chelator agent). Conversely, proteinase K, trypsin, and sodium dodecyl sulfate (SDS) significantly diminished the fungal aggregation. Collectively, our results demonstrated that the aggregation ability of these opportunistic yeast pathogens is time-dependent, and surface proteins and hydrophobic interactions seem to mediate cell aggregation since the presence of proteases and anionic detergents affected the aggregation capability. However, further studies are necessary to better elucidate the molecular aspects of this intriguing phenomenon.

Emergence and circulation of azole-resistant C. albicans, C. auris and C. parapsilosis bloodstream isolates carrying Y132F, K143R or T220L Erg11p substitutions in Colombia.

Methods: Over a four-year period, 123 Candida bloodstream isolates were collected at a quaternary care hospital. The isolates were identified by MALDI-TOF MS and their fluconazole (FLC) susceptibility patterns were assessed according to CLSI guidelines. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pump activity were performed for resistant isolates. Results: Out of 123 clinical strains,C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%; in addition, a high proportion of isolates were cross-resistant to voriconazole. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R, or T220L) were found in 11/19 (58%) of FLCresistant isolates. Furthermore, novel mutations were found in all genes evaluated. Regarding efflux pumps, 8/19 (42%) of FLC-resistant Candida spp strains showed significant efflux activity. Finally, 6/19 (31%) of FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Among FLC-resistant species, C. auris 7/10 (70%) and C. parapsilosis 6/24 (25%) displayed the highest percentages of resistance (C. albicans 6/46, 13%). Discussion: Overall, 68% of FLC-resistant isolates exhibited a mechanism that could explain their phenotype (e.g. mutations, efflux pump activity, or both). We provide evidence that isolates from patients admitted to a Colombian hospital harbor amino acid substitutions related to resistance to one of the most commonly used molecules in the hospital setting, with Y132F being the most frequently detected.

The Mortality Attributable to Candidemia in C. auris Is Higher than That in Other Candida Species: Myth or Reality?

Candida auris has become a major health threat due to its transmissibility, multidrug resistance and severe outcomes. In a case-control design, 74 hospitalised patients with candidemia were enrolled. In total, 22 cases (29.7%) and 52 controls (C. albicans, 21.6%; C. parapsilosis, 21.6%; C. tropicalis, 21.6%; C. glabrata, 1.4%) were included and analysed in this study. Risk factors, clinical and microbiological characteristics and outcomes of patients with C. auris and non-auris Candida species (NACS) candidemia were compared. Previous fluconazole exposure was significantly higher in C. auris candidemia patients (OR 3.3; 1.15-9.5). Most C. auris isolates were resistant to fluconazole (86.3%) and amphotericin B (59%) whilst NACS isolates were generally susceptible. No isolates resistant to echinocandins were detected. The average time to start antifungal therapy was 3.6 days. Sixty-three (85.1%) patients received adequate antifungal therapy, without significant differences between the two groups. The crude mortality at 30 and 90 days of candidemia was up to 37.8% and 40.5%, respectively. However, there was no difference in mortality both at 30 and 90 days between the group with candidemia by C. auris (31.8%) and by NACS (42.3%) (OR 0.6; 95% IC 0.24-1.97) and 36.4% and 42.3% (0.77; 0.27-2.1), respectively. In this study, mortality due to candidemia between C. auris and NACS was similar. Appropriate antifungal therapy in both groups may have contributed to finding no differences in outcomes.

Biomarkers for the severity of periodontal disease in patients with obstructive sleep apnea:IL-1 ß, IL-6, IL-17A, and IL-33.

Objective: This study aims to compare the salivary and gingival crevicular fluid (GCF) concentrations of five cytokines: IL-1ß, IL-6, IL-17A, IL-33, and Tumor Necrosis Factor-alpha (TNF-α) in patients with OSA and their association with periodontitis. Methods: Samples of saliva and GCF were obtained from 84 patients classified into four groups according to periodontal and OSA diagnosis: G1(H) healthy patients, G2(P) periodontitis and non-OSA patients, G3(OSA) OSA and non-periodontitis patients, and G4(P-OSA) periodontitis and OSA patients. The cytokines in the samples were quantified using multiplexed bead immunoassays. Data were analyzed with the Kruskal-Wallis test, Dunn's multiple comparisons test, and the Spearman correlation test. Results: Stage III periodontitis was the highest in patients with severe OSA (69%; p=0.0142). Similar levels of IL-1ß and IL-6 in saliva were noted in G2(P) and G4(P-OSA). The IL-6, IL-17A and IL-33 levels were higher in the GCF of G4(P-OSA). There was a significant positive correlation between IL-33 in saliva and stage IV periodontitis in G4(P-OSA) (r s  = 0.531). The cytokine profile of the patients in G4(P-OSA) with Candida spp. had an increase of the cytokine's levels compared to patients who did not have the yeast. Conclusions: OSA may increase the risk of developing periodontitis due to increase of IL-1ß and IL-6 in saliva and IL-6, IL-17A and IL-33 in GCF that share the activation of the osteoclastogenesis. Those cytokines may be considered as biomarkers of OSA and periodontitis.

Cryptic Oral Microbiota: What Is Its Role as Obstructive Sleep Apnea-Related Periodontal Pathogens?

Periodontitis has been commonly linked to periodontopathogens categorized in Socransky's microbial complexes; however, there is a lack of knowledge regarding "other microorganisms" or "cryptic microorganisms", which are rarely thought of as significant oral pathogens and have been neither previously categorized nor connected to illnesses in the oral cavity. This study hypothesized that these cryptic microorganisms could contribute to the modulation of oral microbiota present in health or disease (periodontitis and/or obstructive sleep apnea (OSA) patients). For this purpose, the presence and correlation among these cultivable cryptic oral microorganisms were identified, and their possible role in both conditions was determined. Data from oral samples of individuals with or without periodontitis and with or without OSA were obtained from a previous study. Demographic data, clinical oral characteristics, and genera and species of cultivable cryptic oral microorganisms identified by MALDI-TOF were recorded. The data from 75 participants were analyzed to determine the relative frequencies of cultivable cryptic microorganisms' genera and species, and microbial clusters and correlations tests were performed. According to periodontal condition, dental-biofilm-induced gingivitis in reduced periodontium and stage III periodontitis were found to have the highest diversity of cryptic microorganism species. Based on the experimental condition, these findings showed that there are genera related to disease conditions and others related to healthy conditions, with species that could be related to different chronic diseases being highlighted as periodontitis and OSA comorbidities. The cryptic microorganisms within the oral microbiota of patients with periodontitis and OSA are present as potential pathogens, promoting the development of dysbiotic microbiota and the occurrence of chronic diseases, which have been previously proposed to be common risk factors for periodontitis and OSA. Understanding the function of possible pathogens in the oral microbiota will require more research.

Patients with obstructive sleep apnea can favor the predisposing factors of periodontitis by the presence of P. melaninogenica and C. albicans, increasing the severity of the periodontal disease.

Objective: The aim of this study was to analyze the cultivable oral microbiota of patients with obstructive sleep apnea (OSA) and its association with the periodontal condition. Methods: The epidemiology profile of patients and their clinical oral characteristics were determined. The microbiota was collected from saliva, subgingival plaque, and gingival sulcus of 93 patients classified into four groups according to the periodontal and clinical diagnosis: Group 1 (n = 25), healthy patients; Group 2 (n = 17), patients with periodontitis and without OSA; Group 3 (n = 19), patients with OSA and without periodontitis; and Group 4 (n = 32), patients with periodontitis and OSA. Microbiological samples were cultured, classified, characterized macroscopically and microscopically, and identified by MALDI-TOF-MS. The distribution of complexes and categories of microorganisms and correlations were established for inter- and intra-group of patients and statistically evaluated using the Spearman r test (p-value <0.5) and a multidimensional grouping analysis. Result: There was no evidence between the severity of OSA and periodontitis (p = 0.2813). However, there is a relationship between the stage of periodontitis and OSA (p = 0.0157), with stage III periodontitis being the one with the highest presence in patients with severe OSA (prevalence of 75%; p = 0.0157), with more cases in men. The greatest distribution of the complexes and categories was found in oral samples of patients with periodontitis and OSA (Group 4 P-OSA); even Candida spp. were more prevalent in these patients. Periodontitis and OSA are associated with comorbidities and oral conditions, and the microorganisms of the orange and red complexes participate in this association. The formation of the dysbiotic biofilm was mainly related to the presence of these complexes in association with Candida spp. Conclusion: Periodontopathogenic bacteria of the orange complex, such as Prevotella melaninogenica, and the yeast Candida albicans, altered the cultivable oral microbiota of patients with periodontitis and OSA in terms of diversity, possibly increasing the severity of periodontal disease. The link between yeasts and periodontopathogenic bacteria could help explain why people with severe OSA have such a high risk of stage III periodontitis. Antimicrobial approaches for treating periodontitis in individuals with OSA could be investigated in vitro using polymicrobial biofilms, according to our findings.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistant Candida glabrata.

BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Pathogenicity Assessment of Colombian Strains of Candida auris in the Galleria mellonella Invertebrate Model.

Candida auris, first described in 2009, is an opportunistic pathogenic yeast that causes nosocomial outbreaks around the world, with high mortality rates associated with therapeutic failure. In this study, we evaluated the pathogenicity of 107 isolates from two cities in Colombia, associated with fungemia or colonization processes; to achieve this, we used the Galleria mellonella invertebrate model to compare pathogenicity. Our results showed that less than half of the total isolates of C. auris presented a high pathogenicity compared to the reference strain SC5314, and most of those highly pathogenic strains were from colonization processes. We observed that there was formation of large aggregates of cells that cannot be disrupted easily, without statistically significant differences between the pathogenicity of the aggregated and non-aggregated strains. In addition, protease activity was observed in 100% of the C. auris strains; phospholipase and hemolysin activity were observed in 67.3 and 68.2% of the studied strains, respectively. In conclusion, these results highlight the utility of determining survival using G. mellonella, which allowed us to provide new information on the pathogenicity, enzymatic activity, and the relationship of the aggregated and non-aggregated phenotypes of C. auris in this model.

Robust, Comprehensive Molecular, and Phenotypical Characterisation of Atypical Candida albicans Clinical Isolates From Bogotá, Colombia.

Candida albicans is commensal in human microbiota and is known to be the commonest opportunistic pathogen, having variable clinical outcomes that can lead to up to 60% mortality. Such wide clinical behaviour can be attributed to its phenotypical plasticity and high genetic diversity. This study characterised 10 Colombian clinical isolates which had already been identified as C. albicans by molecular tests; however, previous bioinformatics analysis of protein mass spectra and phenotypical characteristics has shown that this group of isolates has atypical behaviour, sharing characteristics of both C. africana and C. albicans. This study was aimed at evaluating atypical isolates' pathogenic capability in the Galleria mellonella model; susceptibility profiles were determined and MLST was used for molecular characterisation. Cluster analysis, enabling unbiased bootstrap to classify the isolates and establish their cluster membership and e-BURST, was used for establishing clonal complexes (CC). Both approaches involved using representative MLST data from the 18 traditional C. albicans clades, as well as C. albicans-associated and minor species. Ten atypical isolates were distributed as follows: 6/10 (B71, B41, B60, R6, R41, and R282) were grouped into a statistically well-supported atypical cluster (AC) and constituted a differentiated CC 6; 2/10 of the isolates were clearly grouped in clade 1 and were concurrent in CC 4 (B80, B44). Another 2/10 atypical isolates were grouped in clade 10 and concurred in CC 7 (R425, R111); most atypical isolates were related to geographically distant isolates and some represented new ST. Isolates B41 and R41 in the AC had greater virulence. Isolate B44 was fluconazole-resistant and was grouped in clade 1. The atypical nature of the isolates studied here was demonstrated by the contrast between phenotypical traits (C. africana-like), molecular markers (C. albicans-like), virulence, and antifungal resistance, highlighting the widely described genetic plasticity for this genus. Our results showed that the atypical isolates forming well-differentiated groups belonged to C. albicans. Our findings could contribute towards developing molecular epidemiology approaches for managing hospital-acquired infection.

In-house protocol and performance of MALDI-TOF MS in the early diagnosis of bloodstream infections in a fourth-level hospital in Colombia: Jumping to full use of this technology.

BACKGROUND: Bloodstream infections (BSIs) are a major cause of mortality in hospitalized patients. Rapid diagnosis is crucial because any delay in the antimicrobial treatment is associated with an increase in adverse patient outcomes. The application of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology directly to blood cultures permits earlier identification of BSIs and facilitates treatment management. METHODS: A total of 470 positive blood cultures from patient samples were analyzed using Standard Aerobic/F and Anaerobic/F blood culture media. Isolates were identified using conventional identification methods and by the direct method using the MALDI-TOF MS system. RESULTS: In 470 blood cultures, the direct method showed good identification results (420/470, 89%); specifically, accurate species and genus identification in 283/470 (60%), and only correct genus identification in 137/470 (29%). The direct protocol had better performance for Gram-negative compared to Gram-positive bacteria (97% vs 76%) and was unable to identify the positive blood cultures for both yeasts and some bacteria, mostly Gram-positive (50/470). CONCLUSIONS: The protocol used here gave good and reliable results, being available up to 24 h earlier, while also leading to better use of MALDI-TOF.

Pathogenicity Levels of Colombian Strains of Candida auris and Brazilian Strains of Candida haemulonii Species Complex in Both Murine and Galleria mellonella Experimental Models.

Candida auris and Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) are phylogenetically related species that share some physiological features and habits. In the present study, we compared the virulence of these yeast species using two different experimental models: (i) Galleria mellonella larvae to evaluate the survival rate, fungal burden, histopathology and phagocytosis index and (ii) BALB/c mice to evaluate the survival. In addition, the fungal capacity to form biofilm over an inert surface was analyzed. Our results showed that in both experimental models, the animal survival rate was lower when infected with C. auris strains than the C. haemulonii species complex. The hemocytes of G. mellonella showed a significantly reduced ability to phagocytize the most virulent strains forming the C. haemulonii species complex. Interestingly, for C. auris, it was impossible to measure the phagocytosis index due to a general lysis of the hemocytes. Moreover, it was observed a greater capability of biofilm formation by C. auris compared to C. haemulonii species complex. In conclusion, we observed that C. auris and C. haemulonii complex have different levels of pathogenicity in the experimental models employed in the present study.

Persistence of Clonal Azole-Resistant Isolates of Candida albicans from a Patient with Chronic Mucocutaneous Candidiasis in Colombia.

PURPOSE: The present article describes retrospectively a case of a patient with chronic mucocutaneous candidiasis (CMC) who presented recurrent Candida albicans infection since he was 6 months old. We obtained 16 isolates recovered during a 4-year period. Our purpose was to determinate the susceptibility, genotyping, and the pathogenicity profile in all the isolates. METHODS: Sixteen C. albicans were isolated from a 25-year-old male with several recurrent fungal infections admitted to Hospital. The isolates were recovered during 4 years from a different anatomical origin. We typified them by multilocus sequence typing, also we evaluated susceptibility to fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and amphotericin B by microdilution method and we also test the pathogenic capacity in the Galleria mellonella model. RESULTS: Genotyping of all clinical isolates showed the persistence of the same diploid sequence type (DST). Isolates changed their susceptibility profile over time, but there were no significant statistical differences in pathogenicity. CONCLUSION: Herein, a persistent clonal isolates of C. albicans (DST 918) in a patient with CMC, showed changes in its susceptibility profile after several antifungal treatments acquiring gradual resistance to the azole drugs, which did not affect their pathogenicity.

Development of Amphotericin B Micellar Formulations Based on Copolymers of Poly(ethylene glycol) and Poly(ε-caprolactone) Conjugated with Retinol.

Amphotericin B (AmB) is a broad spectrum of antifungal drug used to treat antifungal diseases. However, due to the high toxicity of AmB, treated patients may suffer the risk of side effects, such as renal failure. Nanoencapsulation strategies have been reported to elicit low toxicity, albeit most of them possess low encapsulation efficiency. The aim of this research is to develop micellar delivery systems for AmB with reduced toxicity while maintaining its affectivity by employing retinol (RET)-conjugated amphiphilic block copolymers (ABCs) as precursors. Copolymers composed of poly(ε-caprolactone) (A) and polyethylenglycol (B) of types AB and ABA were synthesized by ring opening polymerization and subsequently conjugated with RET by Steglich esterification. 1H-NMR spectroscopy was used to corroborate the structure of copolymers and their conjugates and determine their molecular weights. Analysis by gel permeation chromatography also found that the materials have narrow distributions. The resulting copolymers were used as precursors for delivery systems of AmB, thus reducing its aggregation and consequently causing a low haemolytic effect. Upon conjugation with RET, the encapsulation capacity was enhanced from approximately 2 wt % for AB and ABA copolymers to 10 wt %. AmB encapsulated in polymer micelles presented improved antifungal efficiency against Candida albicans and Candida auris strains compared with Fungizone®, as deduced from the low minimum inhibitory concentration.

Impact of calmodulin inhibition by fluphenazine on susceptibility, biofilm formation and pathogenicity of caspofungin-resistant Candida glabrata.

BACKGROUND: In recent decades, Candida glabrata has emerged as a frequent cause of life-threatening fungal infection. In C. glabrata, echinocandin resistance is associated with mutations in FKS1/FKS2 (ß-1,3-glucan synthase). The calmodulin/calcineurin pathway is implicated in response to antifungal stress and calcineurin gene disruption specifically reverses Fks2-mediated resistance of clinical isolates. OBJECTIVES: We evaluated the impact of calmodulin inhibition by fluphenazine in two caspofungin-resistant C. glabrata isolates. METHODS: C. glabrata isolates were identified by ITS1/ITS4 (where ITS stands for internal transcribed spacer) sequencing and the echinocandin target FKS1/FKS2 genes were sequenced. Susceptibility testing of caspofungin in the presence of fluphenazine was performed by a modified CLSI microbroth dilution method. The effect of the fluphenazine/caspofungin combination on heat stress (37°C or 40°C), oxidative stress (0.2 and 0.4 mM menadione) and biofilm formation (polyurethane catheter) was analysed. A Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of this combination on larval survival. RESULTS: F659del was found in the FKS2 gene of both resistant strains. In these clinical isolates, fluphenazine increased susceptibility to caspofungin and reduced their thermotolerance. Furthermore, the fluphenazine/caspofungin combination significantly impaired biofilm formation in an in vitro polyurethane catheter model. All these features participated in the increasing survival of infected G. mellonella after combination treatment in comparison with caspofungin alone. CONCLUSIONS: In a repurposing strategy, our findings confirm that calmodulin could provide a relevant target in life-threatening fungal infectious diseases.

Colombian consensus on the diagnosis, treatment, and prevention of Candida Spp. disease in children and adults, / Consenso Colombiano Para el Diagnóstico, Tratamiento y Prevención de la Enfermedad por Candida Spp. en Niños y Adultos

Invasive Candidiasis (IC) and candidemia (as its most frequent manifestation) have become the main cause of opportunistic mycosis at hospital settings. This study, made by members of the Colombian Association of Infectious Diseases (ACIN), was aimed at providing a set of recommendations for the management, follow-up and prevention of IC / candidemia and mucous membrane candida infection in adult, pediatric and neonatal patients in a hospital setting, including the hemato-oncological and critical care units. All the data obtained through an exhaustive search were reviewed and analyzed in a comprehensive manner by all the members of the group, and the recommendations issued are being made after a careful review of the scientific literature available and the consensus of all specialists involved; the emergence of Candida Spp. problem is highlighted and a correct orientation to health professionals regarding the management of patients with candidiasis is provided in a rational and practical way, emphasizing patient evaluation, diagnostic strategies, prophylaxis, empirical treatment, directed treatment and preventative therapy.

La Candidiasis Invasora (CI) y la candidemia, como su manifestación más frecuente, se ha convertido en la principal causa de micosis oportunista a nivel hospitalario. Este manuscrito realizado por miembros de la Asociación Colombiana de Infectología (ACIN), tuvo como objetivo proporcionar un conjunto de recomendaciones para manejo, seguimiento y prevención de la CI/candidemia y de la infección candidiásica de mucosas, en población adulta, pediátrica y neonatal, en un entorno hospitalario, incluyendo las unidades hemato-oncológicas y unidades de cuidado crítico. Todos los datos obtenidos mediante una búsqueda exhaustiva, fueron revisados y analizados de manera amplia por todos los miembros del grupo, y las recomendaciones emitidas se elaboraron luego de la evaluación de la literatura científica disponible, y el consenso de todos los especialistas involucrados, reconociendo el problema de la emergencia de las infecciones por Candida Spp. y brindando una correcta orientación a los profesionales de la salud sobre el manejo de pacientes con enfermedad candidiásica, de una forma racional y práctica, enfatizando en la evaluación del paciente, estrategias de diagnóstico, profilaxis, tratamiento empírico, tratamiento dirigido y terapia preventiva.

Comparison between MALDI-TOF MS and MicroScan in the identification of emerging and multidrug resistant yeasts in a fourth-level hospital in Bogotá, Colombia.

BACKGROUND: The introduction of MALDI-TOF MS in the clinical microbiology laboratory has modified the approaches for the identification of fungi. Thanks to this tool, it is possible to identify cryptic species, which possess critical susceptibility patterns. Clinical strains were identified using the MicroScan and MALDI-TOF MS systems. Discrepant results from both methods were investigated using ITS rDNA barcoding. Finally, these isolates were also tested for in vitro susceptibility. RESULTS: The percentage of agreement between both methods to 498 yeast isolates was of 93.6% (32 discrepant isolates). The concordance of ITS sequencing with MALDI-TOF MS was higher (99%) than that of MicroScan (94%). Several of these discordant yeasts displayed high MICs for antifungal agents. CONCLUSIONS: Our study highlights the need of the MS and molecular approaches such as MALDI-TOF MS and ITS rDNA barcoding for the correct identification of emerging or cryptic yeast species; besides, some of these could be multidrug resistant. This work was the first experience in the implementation of the MALDI-TOF MS technology in Colombia. We found the first uncommon yeasts including Candida auris and we could identify Trichosporon faecalis. Our work highlights a clear necessity of an accurate yeast identification as a much more pertinent technique than the susceptibility profiles, because the most unusual yeasts exhibit resistance profiles to the few available antifungals.

Microbiota and skin defense peptides may facilitate coexistence of two sympatric Andean frog species with a lethal pathogen.

Management of hyper-virulent generalist pathogens is an emergent global challenge, yet for most disease systems we lack a basic understanding as to why some host species suffer mass mortalities, while others resist epizootics. We studied two sympatric species of frogs from the Colombian Andes, which coexist with the amphibian pathogen Batrachochytrium dendrobatidis (Bd), to understand why some species did not succumb to the infection. We found high Bd prevalence in juveniles for both species, yet infection intensities remained low. We also found that bacterial community composition and host defense peptides are specific to amphibian life stages. We detected abundant Bd-inhibitory skin bacteria across life stages and Bd-inhibitory defense peptides post-metamorphosis in both species. Bd-inhibitory bacteria were proportionally more abundant in adults of both species than in earlier developmental stages. We tested for activity of peptides against the skin microbiota and found that in general peptides did not negatively affect bacterial growth and in some instances facilitated growth. Our results suggest that symbiotic bacteria and antimicrobial peptides may be co-selected for, and that together they contribute to the ability of Andean amphibian species to coexist with the global pandemic lineage of Bd.

Concordance analysis between different methodologies used for identification of oral isolates of Candida species.

BACKGROUND: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. METHODS: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. RESULTS: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. CONCLUSION: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

INTRODUCCIÓN: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. MÉTODOS: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX' Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. RESULTADOS: La PCR Panfungal y secuenciación diferenció 12 especies de Candida' los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación' Vitek® MS' Microflex® y API® 20 C AUX' concordancias agrupadas en las categorías buena y muy buena (wK 0.62 - 0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56 - 0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. CONCLUSIÓN: Los métodos evaluados presentaron una alta concordancia en sus resultados' siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.