PD1 CD44 antiviral peptide as an inhibitor of the protein-protein interaction in dengue virus invasion.

Dengue virus (DENV) infection is mediated by the interaction between the virus envelope protein and cellular receptors of the host cells. In this study, we designed peptides to inhibit protein-protein interaction between dengue virus and CD44 receptor, which is one of the receptors used by DENV for entry. In silico model complexes were designed between domain III of the viral envelope protein of dengue virus 2 and the domain of human CD44 receptor using ClusPro 2.0, (https://cluspro.bu.edu/login.php), and inhibition peptides were designed with Rosetta Online-Server(http://rosie.rosettacommons.org/peptiderive). We identified one linear antiviral peptide of 18 amino acids derived from the human CD44 receptor, PD1 CD44. It did not show hemolysis or toxicity in HepG2 or BHK cell lines, nor did it stimulate the release of IL-1ß, IL-6, TNF-α, and IFN-γ, below 100 µM. It had an IC50 of 13.8 µM and maximum effective dose of 54.9 µM evaluated in BHK cells. The decrease in plaque-forming units/mL for DENV1, DENV2, DENV3, and DENV4 was 99.60%, 99.40%, 97.80%, and 70.50%, respectively, and similar results were obtained by RT-qPCR. Non-structural protein 1 release was decreased in pre- and co-treatment but not in post-treatment. Competition assays between the DN59 peptide, envelope protein, and the fragment of domain III "MDKLQLKGMSYSMCTGKF" of the viral envelope of DENV2 and PD1 CD44 showed that our peptide lost its antiviral activity. We demonstrated that our peptide decreased endosome formation, and we propose that it binds to the envelope protein of DENV, inhibiting viral invasion/fusion.

In Vitro Inhibition of Replication of Dengue Virus Serotypes 1-4 by siRNAs Bound to Non-Toxic Liposomes.

Dengue virus is a ssRNA+ flavivirus, which produces the dengue disease in humans. Currently, no specific treatment exists. siRNAs regulate gene expression and have been used systematically to silence viral genomes; however, they require controlled release. Liposomes show favorable results encapsulating siRNA for gene silencing. The objective herein was to design and evaluate in vitro siRNAs bound to liposomes that inhibit DENV replication. siRNAs were designed against DENV1-4 from conserved regions using siDirect2.0 and Web-BLOCK-iT™ RNAiDesigner; the initial in vitro evaluation was carried out through transfection into HepG2 cells. siRNA with silencing capacity was encapsulated in liposomes composed of D-Lin-MC3-DMA, DSPC, Chol. Cytotoxicity, hemolysis, pro-inflammatory cytokine release and antiviral activity were evaluated using plaque assay and RT-qPCR. A working concentration of siRNA was established at 40 nM. siRNA1, siRNA2, siRNA3.1, and siRNA4 were encapsulated in liposomes, and their siRNA delivery through liposomes led to a statistically significant decrease in viral titers, yielded no cytotoxicity or hemolysis and did not stimulate release of pro-inflammatory cytokines. Finally, liposomes were designed with siRNA against DENV, which proved to be safe in vitro.

Perfil de resistencia genotípica y fenotípica presente en bacterias aisladas a partir de fómites en Armenia, Quindío-Colombia período junio-julio 2019 / Genotypic and phenotypic resistance profile present in bacteria isolated from fomites in Armenia, Quindío-Colombia period June-July 2019

Resumen Introducción: La antibiótico-resistencia es un fenómeno por el cual las bacterias logran sobrevivir al tratamiento con antimicrobianos; con incidencia en ambientes intra y extrahospitalarios como: fuentes hídricas, sector agrario/ganadero y fómites. Objetivo: Describir bacterias presentes en fómites de alta circulación en una región centro-occidental de Colombia junto a su perfil de sensibilidad fenotípica y presencia de genes para betalactamasas tipo TEM-full, OXA-3 y SHV-full. Metodología: Se aislaron cepas bacterianas de billetes, pasamanos de escaleras eléctricas y botones de cajeros automáticos; se evaluó su perfil de sensibilidad fenotípica por medio de concentración mínima inhibitoria-técnica automatizada/Vitek2® y genes para betalactamasas tipo TEM-full, OXA-3 y SHV-full mediante PCR convencional. Resultados: Se obtuvo 30 aislados; Acinetobacter baumannii complex, fue la más común; el fómite con mayor aislados y resistencia fueron los billetes; el 53% portó al menos uno de los genes estudiados. Se identificaron bacterias gramnegativas con resistencia frente a: Imipinem, Piperacilina/Tazobactam, Colistina, Ceftazidima, Tigeciclina y Ceftriaxona; bacterias grampositivas con resistencia frente a: Quinupristina/Dalfopristina, Minociclina, Tetraciclina, Teicoplanina, Nitrofuratoina, Oxacilina, Clindamicina, Trimetropina-sulfametoxazol, y Minociclina. Conclusión: Teniendo en cuenta la circulación de cepas con estas resistencias, es importante la educación en la comunidad para evitar la adquisición o propagación de infecciones por manipulación inadecuada de fómites.

Abstract Introduction: Antibiotic-resistance is a phenomenon by which bacteria manage to survive antimicrobial treatment; with incidence in intra and extra hospital environments such as: water sources, agricultural / livestock sector and fomites. Aim: To describe bacteria present in high circulation fomites in a central-western region of Colombia, with their phenotypic sensitivity profile and presence of genes beta-lactamases (TEM, OXA3 and SHV). Methodology: We isolate bacterial strains from banknotes, escalator handrails and ATM buttons. We evaluated its phenotypic sensitivity profile by minimal inhibitory concentration automated technique using Vitek 2® and presence of genes for beta-lactamases type TEM-full, OXA-3 and SHV-full by conventional PCR. Results: A total of 30 isolates were obtained; Acinetobacter baumannii complex, was the most common; banknotes were the form with the highest number of isolates and resistance. Of the total isolates, 53% carried at least one of the genes studied. Phenotypically, gram-negative bacteria were identified with resistance against: Imipinem, Piperacillin / Tazobactam, Colistin, Ceftazidime, Tigecycline and Ceftriaxone; Gram-positive bacteria with resistance to: Quinupristin / Dalfopristin, Minocycline, Tetracycline, Teicoplanin, Nitrofuratoin, Oxacillin, Clindamycin, Trimethropine-sulfamethoxazole, and Minocycline. Conclusion: Taking into account the circulation of strains with these resistances, it is important to educate the community to avoid the acquisition or spread of infections due to the inappropriate handling of this type of inanimate elements.

Standardization of a latex agglutination test for coproantigen detection of Fasciola sp. in bovine cattle stool.

Fasciolosis is a zoonotic parasitic disease, which affects humans and animals; diagnosed through noncommercial immunoassay tests that cannot be used on the field. Thereby, establishing the optimal conditions to develop a latex agglutination technique with IgG and IgM antibodies directed against excretion/secretion antigens of Fasciola sp. is a priority. Latex particles were sensitized with IgG and IgM antibodies directed against excretion/secretion antigens of Fasciola sp. The specificity of the antibodies was determined against antigens of different helminths and protozoa; the sensitivity and specificity of the test was evaluated against a previously standardized direct ELISA. The coupling rates of the IgG and IgM antibodies were 85.77 and 100%, respectively. The minimum detectable concentration of Fasciola sp. excretion/secretion antigens, diluted in a phosphate-buffered saline, was 1.589 mg/mL(IgG) and 0.158 mg/mL(IgM) and for the antigens incorporated in the bovine cattle stool it was 3.178 mg/mL(IgG) and 1.589 mg/mL(IgM). The test showed crossed reaction against Giardia sp., and Cryptosporidium sp. antigens. Agreement of the IgG and IgM latex test against the ELISA test was of 78.78 and 96.96%, respectively; the specificity found was of 100% for both tests and sensitivity was 78.79% (IgG) and 96.97% (IgM). This work standardized the latex agglutination technique to detect Fasciola sp. antigens in bovine cattle stool.

Manifestaciones clínicas y hallazgos de laboratorio de una serie de casos febriles agudos con diagnóstico presuntivo de infección por el virus dengue. Quindío (Colombia) / Clinical manifestations and laboratory findings on a case series of acute febrile syndrome with a presumptive diagnosis of dengue virus infection. Quindio, Colombia

Objetivo: Describir las manifestaciones clínicas y hallazgos de laboratorio de una serie de casos febriles agudos con diagnóstico presuntivo de infección por el virus dengue. en Quindío (Colombia). Materiales y métodos: Se realizó un estudio de corte transversal, en pacientes con sospecha clínica de dengue en el periodo comprendido entre enero y agosto de 2013, en algunos centros hospitalarios del departamento del Quindío. Se tomaron muestras de sangre para diagnóstico de dengue, leptospira, malaria, hepatitis B, y rickettsiosis. Como pruebas confirmatorias para dengue se realizó aislamiento viral en células C6/36HT y serotipificación para dengue por RTPCR; pruebas de función hepática, cuadro hemático y niveles de citocinas. Resultados: Se caracterizaron 149 casos, de los cuales el 43% presentaron infección por dengue, 4% leptospira, 6,8% rickettsias, un caso de malaria y uno de hepatitis B. En 5 casos se logró el aislamiento del DENV2 y DENV3. Mediante la RT-PCR, se evidenció cocirculación de serotipos 2, 3, 4. Se encontró que las enzimas AST/ALT, el conteo de plaquetas, la erupción y el dolor abdominal fueron marcadores característicos de la infección por dengue, mientras la ictericia y el dolor lumbar se correlacionaron con la leptospirosis. Los valores de citocinas mostraron que la IL-10, TNF α variaron significativamente en casos con dengue frente a otros diagnósticos, y la IL-17 a presentó diferencias significativas en individuos con dengue grave. Conclusiones: El dengue se confirmó como causa etiológica importante de síndrome febril icterohemorrágico en el departamento del Quindío, pero la leptospirosis y la rickettsiosis tienen también una participación importante. Sin embargo, en el 44% de los casos fueron catalogados como síndrome febril indeterminado.

Objective: To characterise the clinical and laboratory findings on a series of febrile cases with a presumptive diagnosis of dengue virus infection in Quindío, Colombia. Materials and methods: This study was conducted from January to July 2013. Blood samples were obtained from patients suspected of dengue virus infection from Quindío department hospitals. These samples were tested for dengue, leptospira, malaria, hepatitis B and rickettsiosis. To confirm dengue infection, we performed viral isolation in C6/36HT cells and dengue serotyping by RT-PCR; liver function tests, complete blood counts and cytokine levels. Results: Of 149 cases, 43% were infected by dengue, 4% leptospira, 6.8% rickettsia, one case of malaria and one case of hepatitis b. We obtained 5 clinical isolates of DENV2 and DENV3 that evidenced co-circulation of serotypes 2, 3, and 4. We found that AST/ALT levels, platelet count, rash and abdominal pain were good markers of infection by dengue, while jaundice and lumbar pain suggested leptospirosis. Cytokine levels revealed that IL-10, TNF a varied significantly in dengue compared with other diagnostics and that IL-17 α showed significant differences in individuals with severe dengue. Conclusions: Dengue was confirmed as an important aetiology of acute febrile icterohaemorrhagic syndrome in Quindío, but leptospirosis and rickettsia also play an important role. However, 44% of the cases were classified as undetermined febrile syndrome.