Análisis de bis(metiltio)gliotoxina como biomarcador para el diagnóstico de Aspergilosis Invasora. Validez de su detección serológica y distribución dentro del género Aspergillus

Tesis llevada a cabo para conseguir el Grado de Doctor en Ciencias por la Universidad de Zaragoza, en junio de 2017.-- Sobresaliente "cum laude".La aspergilosis invasora (AI) es una enfermedad grave, con una elevada morbimortalidad, de la que tienen lugar cada año 200.000 casos aproximadamente. Su diagnóstico precoz es difícil ya que no existe una única prueba que por sí sola dé lugar a un diagnóstico de certeza. Por ello, es necesario evaluar nuevos marcadores, con el fin de conocer la estrategia que permita el diagnóstico más precoz y de la forma más sensible y específica y que sirva para guiar las decisiones terapéuticas. En los últimos años ha aumentado el interés en los productos del metabolismo secundario de Aspergillus spp debido a su potencial utilidad como biomarcadores de AI. Uno de estos metabolitos es la bis(metiltio)gliotoxina (bmGT). Se conoce que diferentes especies de Aspergillus son capaces de producir bmGT. Además, puede detectarse en fluidos biológicos de pacientes en riesgo de AI de forma consistente. Por ello, el objetivo de nuestro trabajo ha sido conocer la distribución de especies fúngicas capaces de producir bmGT y evaluar el rendimiento de la bmGT en el diagnóstico de AI. Nuestros resultados indican que la bmGT podría ser un biomarcador específico de AI ya que no la producen otros géneros distintos de Aspergillus en cultivo líquido. Además, el análisis de sueros de pacientes con fungemia probada, muestra que la probabilidad de reactividad cruzada debida a otros géneros es remota y probablemente no influya en el rendimiento diagnóstico de la bmGT, el cual ha resultado ser mejor que el del galactomanano (GM), uno de los biomarcadores más utilizados en la práctica clínica.Este trabajo ha sido financiado por el Ministerio de Economía y Competitividad y el Fondo Europeo de Desarrollo Regional (FEDER) a través de un contrato de Investigador Iniciado en la Universidad de Zaragoza (PUI15/168), así como por el Instituto de Salud Carlos III a través de un contrato de formación en investigación Río Hortega (CM16/00236), siendo Matxalen Vidal García la beneficiaria de ambos.Peer reviewe

Preparations for Invasion: Modulation of Host Lung Immunity During Pulmonary Aspergillosis by Gliotoxin and Other Fungal Secondary Metabolites

Pulmonary aspergillosis is a severe infectious disease caused by some members of the Aspergillus genus, that affects immunocompetent as well as immunocompromised patients. Among the different disease forms, Invasive Aspergillosis is the one causing the highest mortality, mainly, although not exclusively, affecting neutropenic patients. This genus is very well known by humans, since different sectors like pharmaceutical or food industry have taken advantage of the biological activity of some molecules synthetized by the fungus, known as secondary metabolites, including statins, antibiotics, fermentative compounds or colorants among others. However, during infection, in response to a hostile host environment, the fungal secondary metabolism is activated, producing different virulence factors to increase its survival chances. Some of these factors also contribute to fungal dissemination and invasion of adjacent and distant organs. Among the different secondary metabolites produced by Aspergillus spp. Gliotoxin (GT) is the best known and better characterized virulence factor. It is able to generate reactive oxygen species (ROS) due to the disulfide bridge present in its structure. It also presents immunosuppressive activity related with its ability to kill mammalian cells and/or inactivate critical immune signaling pathways like NFkB. In this comprehensive review, we will briefly give an overview of the lung immune response against Aspergillus as a preface to analyse the effect of different secondary metabolites on the host immune response, with a special attention to GT. We will discuss the results reported in the literature on the context of the animal models employed to analyse the role of GT as virulence factor, which is expected to greatly depend on the immune status of the host: why should you hide when nobody is seeking for you? Finally, GT immunosuppressive activity will be related with different human diseases predisposing to invasive aspergillosis in order to have a global view on the potential of GT to be used as a target to treat IA

In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study

BackgroundIbrexafungerp (SCY-078) is the newest oral and intravenous antifungal drug with broad activity, currently undergoing clinical trials for invasive candidiasis. ObjectiveThe aim of this study was to assess the in vitro activity of ibrexafungerp and comparators against a collection of 434 European blood isolates of Candida. MethodsIbrexafungerp, caspofungin, fluconazole, and micafungin minimum inhibitory concentrations (MICs) were collected from 12 European laboratories for 434 blood isolates, including 163 Candida albicans, 108 Candida parapsilosis, 60 Candida glabrata, 40 Candida tropicalis, 29 Candida krusei, 20 Candida orthopsilosis, 6 Candida guilliermondii, 2 Candida famata, 2 Candida lusitaniae, and 1 isolate each of Candida bracarensis, Candida catenulata, Candida dubliniensis, and Candida kefyr. MICs were determined by the EUCAST broth microdilution method, and isolates were classified according to recommended clinical breakpoints and epidemiological cutoffs. Additionally, 22 Candida auris from different clinical specimens were evaluated. ResultsIbrexafungerp MICs ranged from 0.016 to >= 8 mg/L. The lowest ibrexafungerp MICs were observed for C. albicans (geometric MIC 0.062 mg/L, MIC range 0.016-0.5 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (geometric MIC 0.517 mg/L, MIC range 0.06->= 8 mg/L). Modal MICs/MIC(50)s (mg/L) against Candida spp. were 0.125/0.06 for C. albicans, 0.5/0.5 for C. parapsilosis, 0.25/0.25 for C. glabrata, 0.5/0.5 for C. tropicalis, 1/1 for C. krusei, 4/2 for C. orthopsilosis, and 0.5/0.5 for C. auris. Ibrexafungerp showed activity against fluconazole- and echinocandin-resistant isolates. If adopting wild-type upper limits, a non-wild-type phenotype for ibrexafungerp was only observed for 16/434 (3.7%) isolates: 11 (4.6%) C. parapsilosis, 4 (5%) C. glabrata, and 1 (2.5%) C. tropicalis. ConclusionIbrexafungerp showed a potent in vitro activity against Candida.This study received funding from SCYNEXIS. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of the article, or the decision to submit it for publication. CM-A is a recipient of a grant from Fundació n ONCE (Oportunidad al Talento). EE, AG, NJ, CM-A, and GQ have received grant support from Consejerı́a de Educación, Universidades e Investigación del Gobierno Vasco (GIC15 IT-990-16), Fondo de Investigación Sanitaria del Gobierno de España (FIS PI11/00203), and UPV/EHU (UFI 11/25). All authors declare no other competing interests

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Identification of Rare PB2-D701N Mutation from a Patient with Severe Influenza: Contribution of the PB2-D701N Mutation to the Pathogenicity of Human Influenza

Several amino acid changes have been previously implicated in adaptation of avian influenza viruses to human hosts, among them the D701N change in the PB2 polymerase subunit that also is the main determinant of avian virus pathogenesis in animal models. However, previous studies using recombinant viruses did not provide conclusive information of the contribution of this PB2 residue to pathogenicity in human influenza virus strains. We identified this mutation in an A(H1N1)pdm09-like human influenza virus isolated from an infected patient with pneumonia and acute respiratory failure, admitted to the intensive care unit. An exhaustive search has revealed PB2-D701 as a highly conserved position in all available H1N1 human virus sequences in NCBI database, showing a very low prevalence of PB2-D701N change. Presence of PB2-701N amino acid correlates with severe or fatal outcome in those scarce cases with known disease outcome of the infection. In these patients, the residue PB2-701N may contribute to pathogenicity as it was previously reported in humans infected with avian viruses. This study helps to clarify a debate that has arisen regarding the role of PB2-D701N in human influenza virus pathogenicity.Funding. This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness, Plan Nacional de Investigacion Científica, Desarrollo e Innovación Tecnológica (BFU2014-57797-R) and the network Ciber de Enfermedades Respiratorias (CIBERES) to AN.S

Monitorization, separation and quantification of antifungals used for invasive aspergillosis treatment by High Performance Thin Layer Chromatography

17 slides.-- Slideshow of the talk delivered in the International Symposium for High-Performance Thin-Layer Chromatography. HPTLC 2014, 4th-7th July, Lyon (France).Peer reviewe

Production of the Invasive Aspergillosis Biomarker Bis(methylthio)gliotoxin Within the Genus Aspergillus: In Vitro and in Vivo Metabolite Quantification and Genomic Analysis

Gliotoxin (GT) is a fungal secondary metabolite that has attracted great interest due to its high biological activity since it was discovered by the 1930s. An inactive derivative of this molecule, bis(methylthio)gliotoxin (bmGT), has been proposed as an invasive aspergillosis (IA) biomarker. Nevertheless, studies regarding bmGT production among common opportunistic fungi, including the Aspergillus genus, are scarce and sometimes discordant. As previously reported, bmGT is produced from GT by a methyl-transferase, named as GtmA, as a negative feedback regulatory system of GT production. In order to analyze the potential of bmGT detection to enable identification of infections caused by different members of the Aspergillus genus we have assessed bmGT production within the genus Aspergillus, including A, fumigatus, A. niger, A. nidulans, and A. flavus, and its correlation with gtmA presence. In order to validate the relevance of our in vitro findings, we compared bmGT during in vitro culture with the presence of bmGT in sera of patients from whom the Aspergillus spp. were isolated. Our results indicate that most A. fumigatus isolates produce GT and bmGT both in vitro and in vivo. In contrast, A. niger and A. nidulans were not able to produce GT or bmGT, although A. niger produced bmGT from a exogenous GT source. The frequency and amount of bmGT production in A. terreus and A. flavus isolates in vitro was lower than in A. fumigatus. Our results suggest that this defect could be related to the in vitro culture conditions, since isolates that did not produce bmGT in vitro were able to synthetize it in vivo. In summary, our study indicates that bmGT could be very useful to specifically detect the presence of A. fumigatus, the most prevalent agent causing IA. Concerning A. terreus and A. flavus a higher number of analyses from sera from infected patients will be required to reach a useful conclusion

Capacidad de las especies de Aspergillus para metilar gliotoxina y producir el biomarcador de aspergilosis invasivo bis (metiltio) gliotoxina

1 figure.-- Abstract of the Poster presented at the 8th Advaces Against Aspergillosis conference, in Lisbon, (Portugal), from 1st to 3rd February 2018.[EN] [Purpose]: Bis(methylthio)gliotoxin (bmGT) is a novel invasive aspergillosis biomarker which has been recently validated. It is produced from gliotoxin (GT), a virulence factor produced by Aspergillus spp. It is known that A. fumigatus produces GT more frequently and in higher concentration than other Aspergillus species. However, data regarding bmGT production within the genus Aspergillus are scarce. In previous studies we found out that bmGT was detected in culture supernatants from non-A. fumigatus species in low rates. In this study we assess the ability to produce bmGT from an exogenous source of GT for non-A. fumigatus species. It is important to understand if all Aspergillus species are able to produce bmGT in order to know the specificity of this invasive aspergillosis biomarker. [Methods]: We assessed a total of 35 clinical isolates of the species A. flavus (n = 12), A. nidulans (n = 6), A. niger (n = 8) and A. terreus (n = 9). A 12 McFarland conidial suspension (approximately 3 – 5 x 107 conidia/mL) was added (1/10) to liquid medium (Czapek Dox Broth + glutamine 2 mM + HEPES 25 mM) in 50 mL culture flasks and incubated at 37 ºC for 45 h. Then, GT was added to a final concentration of 2.5 mg/L and methanol was added as solvent control. At 0, 3 and 6 h, 2 mL aliquots of supernatant were taken and GT and bmGT were quantified by High Performance Thin Layer Chromatography. Statistical analysis and figures were performed with Prism 6, Graphpad, San Diego, CA, USA. [Results]: All isolates from the A. flavus (n = 12) and A. terreus species (n=9) were able to methylate exogenous GT in order to produce bmGT. Among A. niger isolates, this ability was less consistent, nevertheless, 5/8 isolates (62.5 %) showed such ability. Finally, none of the A. nidulans isolates (n = 6) produced bmGT. Among bmGT producing isolates, at 0 h after GT addition, it was recovered in a concentration of 0.86 ± 0.04 mg/L (mean ± standard error of mean). None of the isolates produced bmGT at this time (Figure 1). At 3 h after GT addition, its concentration decreased and bmGT concentration increased. This observation continued at 6 h, when the maximum bmGT and the minimum GT were detected. There were no differences between mean concentration of GT and bmGT among species (Sidak’s multiple comparisons test, p > 0.05). [Conclusion]: BmGT was produced by all the Aspergillus species analysed but A. nidulans. A. flavus and A. terreus consistently produced bmGT (100%) and 62.5 % of the isolates from A. niger were also able to produce it. These data suggest that bmGT can be produced by a high percentage of isolates. Therefore, bmGT could be detected in serum from patients suffering an infection due to bmGT producing isolates. According to our data, this biomarker would be highly specific for infections due to Aspergillus species other than A. nidulans.[ES] [Objetivo]: La bis (metiltio) gliotoxina (bmGT) es un nuevo biomarcador de aspergilosis invasivo que ha sido validado recientemente. Se produce a partir de gliotoxina (GT), un factor de virulencia producido por Aspergillus spp. Se sabe que A. fumigatus produce GT con más frecuencia y en mayor concentración que otras especies de Aspergillus. Sin embargo, los datos sobre la producción de bmGT dentro del género Aspergillus son escasos. En estudios anteriores descubrimos que se detectó bmGT en sobrenadantes de cultivos de especies distintas de A. fumigatus en tasas bajas. En este estudio evaluamos la capacidad de producir bmGT a partir de una fuente exógena de GT para especies distintas de A. fumigatus . Es importante comprender si todosLas especies de Aspergillus son capaces de producir bmGT para conocer la especificidad de este biomarcador invasivo de aspergilosis. [Métodos]: Se evaluaron un total de 35 aislamientos clínicos de las especies A. flavus (n = 12), A. nidulans (n = 6), A. niger (n = 8) y A. terreus (n = 9). Una suspensión 12 McFarland conidial (aproximadamente 3 - 5 x 10 7 conidios / ml) se añadió (1/10) a un medio líquido (Czapek Dox Broth + glutamina 2 mM + HEPES 25 mM) en 50 matraces de cultivo mL y se incubaron a 37 ºC durante 45 h. Luego, se añadió GT hasta una concentración final de 2,5 mg / L y se añadió metanol como control de disolvente. A las 0, 3 y 6 h, se tomaron alícuotas de 2 ml de sobrenadante y se cuantificaron la GT y la bmGT mediante cromatografía de capa fina de alta resolución. El análisis estadístico y las cifras se realizaron con Prism 6, Graphpad, San Diego, CA, EE. UU. [Resultados]: Todos los aislamientos de las especies A. flavus (n = 12) y A. terreus (n = 9) fueron capaces de metilar GT exógena para producir bmGT. Entre los aislamientos de A. niger , esta habilidad fue menos consistente, sin embargo, 5/8 aislamientos (62.5%) mostraron tal habilidad. Finalmente, ninguno de los aislados de A. nidulans (n = 6) produjo bmGT. Entre los aislados productores de bmGT, a las 0 h después de la adición de GT, se recuperó en una concentración de 0,86 ± 0,04 mg / L (media ± error estándar de la media). Ninguno de los aislamientos produjo bmGT en este momento (Figura 1). A las 3 h después de la adición de GT, su concentración disminuyó y la concentración de bmGT aumentó. Esta observación continuó a las 6 h, cuando se detectaron la bmGT máxima y la GT mínima. No hubo diferencias entre la concentración media de GT y bmGT entre las especies (prueba de comparaciones múltiples de Sidak, p> 0,05). [Conclusión]: La BmGT fue producida por todas las especies de Aspergillus analizadas excepto A. nidulans . A. flavus y A. terreus produjeron consistentemente bmGT (100%) y el 62,5% de los aislados de A. niger también pudieron producirla. Estos datos sugieren que la bmGT puede ser producida por un alto porcentaje de aislamientos. Por lo tanto, la bmGT podría detectarse en suero de pacientes que padecen una infección debido a aislamientos productores de bmGT. Según nuestros datos, este biomarcador sería altamente específico para infecciones debidas a especies de Aspergillus distintas de A. nidulans .Peer reviewe