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

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

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)

Anti-Candida Antibodies of Patients with Invasive Candidiasis Inhibit Growth, Alter Cell Wall Structure, and Kill Candida albicans In Vitro

Invasive candidiasis (IC), caused by Candida yeasts, particularly Candida albicans, poses a significant threat with high mortality rates. Diagnosis is challenging due to Candida's common presence in human microbiota. To address this, our research group developed an immunofluorescence assay detecting Candida albicans Germ Tube Antibodies (CAGTA) in IC patients. CAGTA, indicative of invasive processes, is associated with a lower mortality rate in ICU patients. Based on this premise, this study aims to provide results regarding the lack of knowledge about the potential activity of CAGTA against invasive infections in humans caused by the fungus Candida albicans. Therefore, in order to characterize the activity of CAGTA produced by patients with IC, we used sera from 29 patients with IC caused by either C. albicans or non-albicans Candida species. Whole serum IgG antibodies were fractionated into anti-blastospores, CAGTA-enriched, and purified CAGTA and the assessments included XTT colorimetric assays for metabolic activity, CFU counts for viability, and microscopy for growth, viability, and morphological analysis. The CAGTA-enriched IgG fraction significantly reduced the metabolic activity and viability of C. albicans compared to anti-blastospores. Purified CAGTA altered germ tube cell wall surfaces, as revealed by electron microscopy, and exhibited fungicidal properties by DiBAC fluorescent staining. In conclusion, antibodies in response to invasive candidiasis have antifungal activity against Candida albicans, influencing metabolic activity, viability, and cell wall structure, leading to cell death. These findings suggest the potential utility of CAGTA as diagnostic markers and support the possibility of developing immunization protocols against Candida infections.Funding for this work was provided by the University of the Basque Country UPV/EHU (GIU21/017) project. G. C. was supported by a Department of Education, Universities and Research of the Basque Country fellowship (PRE_2013_562). M. B. was supported by a University of Basque Country fellowship (PIF19/316)

Molecular Identification of Fungal Species through Multiplex-qPCR to Determine Candidal Vulvovaginitis and Antifungal Susceptibility

Vulvovaginal candidiasis (VVC) is a prevalent condition affecting women worldwide. This study aimed to develop a rapid qPCR assay for the accurate identification of VVC etiological agents and reduced azole susceptibility. One hundred and twenty nine vaginal samples from an outpatient clinic (Bilbao, Spain) were analyzed using culture-based methods and a multiplex qPCR targeting fungal species, which identified Candida albicans as the predominant species (94.2%). Antifungal susceptibility tests revealed reduced azole susceptibility in three (3.48%) isolates. Molecular analysis identified several mutations in genes associated with azole resistance as well as novel mutations in TAC1 and MRR1 genes. In conclusion, we developed a rapid multiplex qPCR assay that detects C. albicans in vulvovaginal specimens and reported new mutations in resistance-related genes that could contribute to azole resistance.This research was funded by a Basque Government project, number IT 913-16_Basque University System Research Groups (to M.-D.M.), University of the Basque Country project, GIU21/07_Research Groups of the University of the Basque Country UPV/EHU (to Í.F.-d.-L.), and a Collaborative Research Project among UPV/EHU Groups (to M.-D.M., Í.F.-d.-L. and I.A.) P.M.M received a predoctoral grant from the Department of Education, Universities, and Research of the Basque Government (Programa de Formación de Personal Investigador No Doctor)

Candida albicans cDNA library screening reveals novel potential diagnostic targets for invasive candidiasis

The detection of patterns associated with the invasive form of Candida albicans, such as Candida albicans germ tube antibodies (CAGTA), is a useful complement to blood culture for Invasive Candidiasis (IC) diagnosis. As CAGTA are detected by a non-standardisable and non-automatable technique, a Candida albicans cDNA expression library was screened with CAGTA isolated from serum of an animal model of invasive candidiasis, and five protein targets were identified: hyphally regulated cell wall protein 1 (Hyr1), enolase 1 (Eno1), coatomer subunit gamma (Sec21), a metallo-aminopeptidase (Ape2) and cystathionine gamma-lyase (Cys3). Homology with proteins from other organisms rules out Cys3 as a good biomarker while Sec21 results suggest that it is not in the germ tubes surface but secreted to the external environment. Our analysis propose Ape2, Sec21 and a region of Hyr1 different from the one currently being studied for immunoprotection as potential biomarker candidates for the diagnosis of IC.Funding for this work was provided by the University of the Basque Country UPV/EHU (GIU21/017) and the Basque Government (IT913-16). M. B-V was supported by a University of the Basque Country UPV/EHU fellowship (PIF19/316). G. C. and P. M-M were supported by Department of Education, Universities and Research of the Basque Country fellowships (PRE_2013_562; PRE_2017_1_0159)