Identification of superficial Candida albicans germ tube antigens in a rabbit model of disseminated candidiasis. A proteomic approach

The diagnosis of invasive candidiasis remains a clinical challenge. The detection by indirect immunofluorescence of Candida albicans germ-tube-specific antibodies (CAGTA), directed against germ-tube surface antigens, is a useful diagnostic tool that discriminates between colonization and invasion. However, the standardization of this technique is complicated by its reliance on subjective interpretation. In this study, the antigenic recognition pattern of CAGTA throughout experimental invasive candidiasis in a rabbit animal model was determined by means of 2D-PAGE, Western blotting, and tandem mass spectrometry (MS/MS). Seven proteins detected by CAGTA were identified as methionine synthase, inositol-3-phosphate synthase, enolase 1, alcohol dehydrogenase 1,3-phosphoglycerate kinase, 14-3-3 (Bmh1), and Egd2. To our knowledge, this is the first report of antibodies reacting with Bmh1 and Egd2 proteins in an animal model of invasive candidiasis. Although all of the antigens were recognized by CAGTA in cell-wall dithiothreitol extracts of both germ tubes and blastospores of C. albicans, immunoelectron microscopy study revealed their differential location, as the antigens were exposed on the germ-tube cell-wall surface but hidden in the inner layers of the blastospore cell wall. These findings will contribute to developing more sensitive diagnostic methods that enable the earlier detection of invasive candidiasis. [Int Microbiol 2014; 17(1):21-29]Keywords: Candida albicans · germ tube antibodies · invasive candidiasis · rabbit mode

Use of Recombinant Antigens for the Diagnosis of Invasive Candidiasis

Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis

Low Sensitivity of Conventional Fungal Agars in Fungemia by Rhodotorula Mucilaginosa: Description of Two Cases

Background Although most bloodstream yeast infections are caused by Candida spp., infections by rare or less common species have increased in recent years. Diagnosis of infections caused by these species is difficult due to the lack of specific symptoms and adequate diagnostic tools. Cases presentation We describe two cases of fungemia by Rhodotorula mucilaginosa within a few months of each other, in a secondary Spanish hospital. In both cases, diagnosis was challenging. Blood subcultures in conventional fungal media were persistently negatives and the use of non-conventional fungal media was essential for isolating the yeasts and achieving a correct diagnosis. 1-3 beta-d-glucan detection and a panfungal PCR assay were helpful techniques to confirm the diagnosis Conclusion It is highly important to establish an early diagnosis for fungemia. The process is challenging because often non-specific symptoms are presents. When yeasts grow in blood cultures other genera than Candida spp. could be the cause of infection. Patient risk factors should be assessed to incorporate alternative culture media and the available rapid diagnostic test, in order to provide an early recognition of the pathogenThis work was supported by research project PI17CIII/00033 from the Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III, and IT913-16 from the Consejeria de Educacion, Universidades e Investigacion of Gobierno Vasco-Eusko Jaurlaritz

Aislamiento de Candida dubliniensis en un adolescente con estomatitis protésica

Objetivos: Utilizar varios métodos que permiten la diferenciación entre Candida albicans y Candida dubliniensis en un intento de conocer si C. dubliniensis puede ser aislada de la cavidad oral de adolescentes con prótesis ortopédicas orales. Materiales y métodos: Se aislaron 12 cepas de género Candida procedentes de mucosa palatina y de soporte de prótesis de 12 pacientes adolescentes portadores de prótesis ortopédicas orales. Para la diferenciación entre C. albicans y C. dubliniensis se utilizaron varias pruebas fenotípicas (la asimilación de fuentes de carbono con el método comercial ID 32C, el crecimiento en agar glucosado de Sabouraud a 45 ºC, la producción abundante de clamidosporas en agar caseína, y la reactividad mediante inmunofluorescencia indirecta con un antisuero específico para C. dubliniensis) y la reacción en cadena de la polimerasa (PCR). El serotipado de C. albicans se realizó con el anticuerpo monoclonal B9E. Resultados: Los 12 pacientes estudiados presentaron una estomatitis protésica tipo 2 de Newton y en todos los casos se aislaron las mismas especies de la muestra de mucosa y de la de prótesis del mismo paciente. El CHROMagar Candida y la prueba de la filamentación en suero permitieron diferenciar los aislamientos que daban lugar a colonias de color verde y filamentaban de los que daban colonias violetas y no lo hacían. Unicamente el aislamiento del paciente 8 fue positivo con el antisuero específico para C. dubliniensis y produjo abundantes clamidosporas en agar caseína, mientras que ocho aislamientos no presentaron crecimiento a 45 ºC. La identificación de todos los aislamientos se consiguió con la prueba ID 32C, identificándose C. albicans en el 75% de los pacientes, C. glabrata en el 16,6% y C. dubliniensis en el 8,3%. La PCR con iniciadores específicos para el tipado de C. dubliniensis permitió la identificación del aislamiento del paciente 8 como C. dubliniensis genotipo 1. Conclusión: C. dubliniensis puede ser aislada de la cavidad oral de adolescentes con estomatitis asociada a prótesis ortopédicas y es posible, y técnicamente asequible, la diferenciación entre C. albicans y C. dubliniensis mediante la realización de pruebas como el ID 32C, la observación de abundantes clamidosporas en agar caseína, la reactividad con un antisuero específico para C. dubliniensis y la PCR.Objectives: Test several methods that allow the differentiation between Candida albicans and Candida dubliniensis, in an attempt to assess whether C. dubliniensis can be recovered from the oral cavity of teenagers wearing orthopedic oral prostheses. Material and Methods: Twelve Candida strains were isolated from the prosthesis as well as the palatal mucosa in contact with the dental prosthesis from 12 teenager patients wearing orthopedic oral prostheses. Differentiation between C. albicans and C. dubliniensis was achieved by a number of phenotypic tests (carbon assimilation by the commercially available ID 32C test, growth at 45ºC on Sabouraud glucose agar, abundant chlamydospore production on Casein agar, and reactivity with a C. dubliniensis antiserum) and the polymerase chain reaction (PCR). Serotyping of C. albicans was performed with monoclonal antibody B9E. Results: All 12 patients studied presented a Newton's type 2 denture stomatitis and in every patient the same Candida species were isolated from the prosthesis and the palatal mucosa in contact with the dental prosthesis. CHROMagar Candida and the germ tube test allowed the differentiation of isolates giving green colonies and a positive germ tube test from those giving violet colonies and a negative germ tube test. Only the isolate from patient 8 was stained by the C. dubliniensis antiserum and showed abundant chlamydospore production on Casein agar. Eight isolates did not grow at 45 ºC. Identification of all isolates was obtained by the ID 32C test. C. albicans was identified in 75% of patients, C. glabrata in 16,6% and C. dubliniensis in 8,3%. By using specific primers for typing C. dubliniensis, PCR allowed the identification of patient's 8 isolate as C. dubliniensis genotype 1. Conclusion: C. dubliniensis can be isolated from the oral cavity of teenagers wearing orthopedic oral prostheses and it is possible and technically amenable, the differentiation between C. albicans y C. dubliniensis using the ID 32C test, the observation of abundant chlamydospore production on Casein agar, the reactivity with a C. dubliniensis antiserum and the PCR

A novel Candida albicans Als3, Hwp1 and Met6 derived complex peptide protects mice against hematogenously induced candidiasis

Candida albicans can cause superficial or systemic infections in humans, particularly in immunocompromised individuals. Vaccination strategies targeting specific antigens of C. albicans have shown promise in providing protection against invasive candidiasis. This study aimed to evaluate the immuno-protective capacity of a KLH conjugated complex peptide, 3P-KLH, containing epitopes from C. albicans antigens Als3, Hwp1, and Met6 in a murine model of hematogenously induced candidiasis. Mice immunized with 3P-KLH raised a specific antibody response, and protection against C. albicans infection was assessed. Immunized mice exhibited significantly lower fungal load in their kidneys compared to the control group. Moreover, 37.5 % of immunized mice survived 21 days after the infection, while all control animals died within the first nine days. These findings suggest that the 3P-KLH complex peptide, targeting C. albicans key antigens, elicits a protective immune response and reduces the severity of systemic Candida infection. In addition, the high binding affinity of the selected epitopes with MHC II alleles further supports the potential immunogenicity of this peptide in humans. This research provides insights into the development of novel immunotherapeutic approaches against invasive candidiasis.Funding for this work was provided by the University of the Basque Country UPV/EHU (GIU21/017) and the Basque Government (IT913-16). A. D. was supported by Fundación Jesus de Gangoiti Barrera. G. C. was supported by a Department of Education, Universities and Research of the Basque Country fellowship (PRE_2013_562)

Biomarkers for the diagnosis of invasive candidiasis in immunocompetent and immunocompromised patients

Blood culture methods show low sensitivity, so reliable non-culture diagnostic tests are needed to help clinicians with the introduction, de-escalation, and discontinuation of antifungal therapy in patients with suspected invasive candidiasis (IC). We evaluated different biomarkers for the diagnosis of IC in immunocompetent and immunocompromised patients at risk for developing invasive fungal diseases. The specificity of Candida albicans germ-tube antibodies (CAGTA) detection was high (89%-100%), but sensitivity did not exceed 61% even after raising the cut-off from 1/160 to 1/80. We developed enzyme-linked immunoassays detecting antibodies against C. albicans proteins (Als3-N, Hwp1-N, or Met6) that resulted more sensitive (66%-92%) but less specific than CAGTA assay. The combination of 1,3-beta-D-glucan (BDG) detection and CAGTA results provided the highest diagnostic usefulness in immunocompetent patients. However, in immunocompromised patients, anti-Met6 antibodies was the best biomarker, both, alone or in combination with BDGThis work was supported by the Basque Government (Groups of Research IT913-16; GIC15/103). Marta Bregón-Villahoz received a grant from the University of the Basque Country UPV/EHU (PIF19/316)

Antibacterial and antifungal activity of the human endometrial fluid during the natural cycle

[EN] Purpose. Some microbiota patterns have been associated with favorable IVF prognosis and others with pathological conditions. The endometrial fluid aspirate (EFA) contains antibacterial proteins that are enriched in implantative IVF cycles, but the antimicrobial effect of EFA has not been addressed. We aimed to evaluate the antimicrobial activity of the human endometrial fluid during the natural cycle. Methods. EFA was obtained through an embryo transfer catheter in 38 women, aged 18-40 years, with regular cycles attending to a fertility clinic. The antimicrobial activity of EFAs was tested against two strains of Staphylococcus aureus; one strain each of Streptococcus agalactiae, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae; and three yeasts (Candida albicans, Candida glabrata, and Candida krusei). Results. All samples exhibited antibacterial activity against S. aureus. In addition, 32.4% of EFAs were active against one of the other microorganisms assayed, 16.2% against two, and 5.4% against four of them. In contrast, none exhibited antibacterial activity against E. coli or K. pneumoniae. The antimicrobial activity differs considerably between EFA samples, and we failed to observe a cycle-related pattern. Conclusions. EFA presented two antimicrobial activity patterns: (a) one common to all the samples, exhibiting activity against S. aureus and lack of activity against E. coli and K. pneumoniae, and (b) an individualized pattern, showing activity against some of the other microorganisms tested. The intensity of antibacterial activity differs between EFA samples. Our data suggest that the uterine microbiota is controlled by means of endometrial fluid components.This study was partially supported by a Grant for Fertility Innovation (GFI, 2011) from Merck, Darmstadt, Germany. M. Bregón-Villahoz is recipient of a predoctoral grant from the Universidad del País Vasco-Euskal Herriko Unibertsitatea (UPV/EHU) (PIF19/316). The authors thank the technical and human support provided by DNA Bank Service (SGIker) of the University of the Basque Country (UPV/EHU) and European funding (ERDF and ESF). CIC bioGUNE is accredited with the Severo Ochoa Excellence award by the Spanish Ministerio de Economía y Competitividad, MINECO (SEV-2016-0644)

Ultrasonography in rheumatology: time to learn from patient views

Objective: The objective of this observational, descriptive, cross-sectional, multicentre study was to assess the perceived quality and grade of satisfaction expressed by patients with chronic arthropathies regarding the use of musculoskeletal (MSK) ultrasonography by rheumatologists as an integrated clinical care tool. Methods: All Spanish rheumatology departments with MSK ultrasonography incorporated in their healthcare services were invited to participate in the study. A Spanish-language survey was offered to fill out anonymously to all consecutive patients with chronic arthropathies under follow-up in the rheumatology outpatient clinics who attended their centre for a period of 3 months. The survey consisted of three sections. The first section contained patients’ demographics, disease data, frequency of performing rheumatological ultrasound and information about who performed their ultrasound assessments. The second section consisted of 14 questions about patient’s experience and opinion on different aspects of the management, performance and perceived usefulness of performing ultrasound, to be answered on a Likert scale 1–5. The third section of the survey was addressed to the rheumatologist ultrasonographers. Results: Nine hundred and four patients from 16 university hospital rheumatology departments completed the survey. All questions reached an overall favourable response ≥ 80%. Patients who reported usual ultrasound examinations in their rheumatology care and those in which it was their attending rheumatologist who performed the ultrasound assessments responded more favourably. Conclusion: Our encouraging patient-centred results may be useful in facilitating the implementation of rheumatological ultrasound in rheumatology care worldwide. Key Points • This is the largest multicentre survey carried out in patients with chronic joint diseases designed to assess their experience and perceived benefits with the use of ultrasonography performed by rheumatologists in daily practice. • Musculoskeletal ultrasound incorporated into rheumatology care was very well accepted and valued by most patients. • The patients perceived that ultrasonography helps not only their rheumatologist but also themselves to better understand their condition. • The patients believed that ultrasonography helps them accept and comply with the proposed treatmen

Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

Background: Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. the possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal Findings: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. the inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/Significance: the high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. the easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.Department of Education, Universities and Research, Basque GovermentFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Istituto Superiore di Sanita, National Research Project on A.I.D.S.Cariparma Banking FoundationBrazilian National Research CouncilUniv Parma, Sez Microbiol, Dipartimento Patol, I-43100 Parma, ItalyUniv Basque Country, Fac Med Odontol, Dept Inmunol, Microbiol Parasitol, Bilbao, SpainUniv Basque Country, Dept Enfermeria I, Bilbao, SpainUniv Milan, Dipartimento Sci Cliniche L Sacco, Sez Malattie Infettive Immunopatol, Milan, ItalyUniv Studi Parma, Dipartimento Clin Med, Nefrol Sci Prev, Parma, ItalyUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilDepartment of Education, Universities and Research, Basque Goverment: IT-264-07FAPESP: 06/50634-2Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 50G.30Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 40D.14Cariparma Banking Foundation: 2004.0190Brazilian National Research Council: research fellowshipWeb of Scienc

Monitoreo inteligente orientado al diagnóstico de fallas en equipos industriales

Consulta en la Biblioteca ETSI Industriales (8877)[ES] La presente tesis tiene como objetivo aplicar un procedimiento de detección de fallas a datos reales de la operación de un reactor de lecho fijo para la hidrogenación de gas natural empleado luego en la producción de metanol en la planta de Repsol-YPF de Plaza Huincul (Neuquén). Asimismo, se formulará un procedimiento de diagnóstico para analizar posibles fallas ocurridas en la operación de esta sección de la planta. Para la detección se hará uso de una de las herramientas más frecuentemente empleadas, que consiste en la descomposición en Componentes Principales, conocida como PCA (Principal Component Analysis), por sus siglas en inglés. Para implementar el procedimiento de cálculo y análisis de datos se utilizará el utilitario MatLab, con el cual se obtendrán los parámetros estadísticos que permiten la visualización de los periodos donde ocurrieron fallas. En cuanto al diagnóstico, se implementará un procedimiento en base a simulación cualitativa que permite inferir las tendencias de variación de distintas variables frente a una dada falla en la operación. Se propondrán dos posibles escenarios de fallas para testear el procedimiento implementado y se comparará con el comportamiento real de la planta durante un periodo durante el cual se haya detectado la posibilidad de una falla.Rius Moragues, MD. (2011). Monitoreo inteligente orientado al diagnóstico de fallas en equipos industriales. http://hdl.handle.net/10251/29265.Archivo delegad