Molecular Diagnosis of Endemic Mycoses

Endemic mycoses; Molecular diagnosisMicosis endémicas; Diagnóstico molecularMicosis endèmiques; Diagnòstic molecularDiagnosis of endemic mycoses is still challenging. The moderated availability of reliable diagnostic methods, the lack of clinical suspicion out of endemic areas and the limitations of conventional techniques result in a late diagnosis that, in turn, delays the implementation of the correct antifungal therapy. In recent years, molecular methods have emerged as promising tools for the rapid diagnosis of endemic mycoses. However, the absence of a consensus among laboratories and the reduced availability of commercial tests compromises the diagnostic effectiveness of these methods. In this review, we summarize the advantages and limitations of molecular methods for the diagnosis of endemic mycoses.This research was funded by research project PI21CIII/00007 from Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III

Dot Immunobinding Assay for the Rapid Serodetection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

Scedosporium; Cystic fibrosis; Serological detectionScedosporium; Fibrosi quística; Detecció serològicaScedosporium; Fibrosis quística; Detección serológicaThe detection of Scedosporium/Lomentospora is still based on non-standardized low-sensitivity culture procedures. This fact is particularly worrying in patients with cystic fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because a poor and delayed diagnosis can worsen the prognosis of the disease. To contribute to the discovery of new diagnostic strategies, a rapid serological dot immunobinding assay (DIA) that allows the detection of serum IgG against Scedosporium/Lomentospora in less than 15 min was developed. A crude protein extract from the conidia and hyphae of Scedosporium boydii was employed as a fungal antigen. The DIA was evaluated using 303 CF serum samples (162 patients) grouped according to the detection of Scedosporium/Lomentospora in the respiratory sample by culture, obtaining a sensitivity and specificity of 90.48% and 79.30%, respectively; positive and negative predictive values of 54.81% and 96.77%, and an efficiency of 81.72%. The clinical factors associated with the results were also studied using a univariate and a multivariate analysis, which showed that Scedosporium/Lomentospora positive sputum, elevated anti-Aspergillus serum IgG and chronic Pseudomonas aeruginosa infection were significantly associated with a positive result in DIA, while Staphylococcus aureus positive sputum showed a negative association. In conclusion, the test developed can offer a complementary, rapid, simple and sensitive method to contribute to the diagnosis of Scedosporium/Lomentospora in patients with CF.This research was funded by the Basque Government, grant numbers IT1362-19 and IT1657-22. L.M-S and M.A have received a predoctoral grant from the Basque Government and L.A-F from the University of the Basque Country (UPV/EHU)

Diagnosis and Stratification of Pseudomonas aeruginosa Infected Patients by Immunochemical Quantitative Determination of Pyocyanin From Clinical Bacterial Isolates

Pseudomonas aeruginosa; Monoclonal antibody; PyocyaninPseudomonas aeruginosa; Anticuerpo monoclonal; PiocianinaPseudomonas aeruginosa; Anticòs monoclonal; PiocianinaThe development of a highly sensitive, specific, and reliable immunochemical assay to detect pyocyanin (PYO), one of the most important virulence factors (VFs) of Pseudomonas aeruginosa, is here reported. The assay uses a high-affinity monoclonal antibody (mAb; C.9.1.9.1.1.2.2.) raised against 1-hydroxyphenazine (1-OHphz) hapten derivatives (PC1; a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids). Selective screening using PYO and 1-OHphz on several cloning cycles allowed the selection of a clone able to detect PYO at low concentration levels. The microplate-based ELISA developed is able to achieve a limit of detection (LoD) of 0.07 nM, which is much lower than the concentrations reported to be found in clinical samples (130 μM in sputa and 2.8 μM in ear secretions). The ELISA has allowed the investigation of the release kinetics of PYO and 1-OHphz (the main metabolite of PYO) of clinical isolates obtained from P. aeruginosa-infected patients and cultured in Mueller-Hinton medium. Significant differences have been found between clinical isolates obtained from patients with an acute or a chronic infection (~6,000 nM vs. ~8 nM of PYO content, respectively) corroborated by the analysis of PYO/1-OHphz levels released by 37 clinical isolates obtained from infected patients at different stages. In all cases, the levels of 1-OHphz were much lower than those of PYO (at the highest levels 6,000 nM vs. 300 nM for PYO vs. 1-OHphz, respectively). The results found point to a real potential of PYO as a biomarker of P. aeruginosa infection and the possibility to use such VF also as a biomarker for patient stratification[2] and for an effective management of these kinds of infections.This work has been funded by the Spanish Government to M-PM through the Ministry of Science and Innovation (SAF2015-67476-R, RTI2018-096278-B-C21, PI, M-PM) and by Fundació Marató de TV3 (201825-30-31, PI, M-PM). The Nb4D group is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient: 2017 SGR 1441). CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund (ERDF). Moreover, BR-U has an FI fellowship from the AGAUR (Agència de Gestió d’Ajuts Universitaris I de Recerca) of the Government of Catalonia (Generalitat de Catalunya) (2019FI_B00289). El Fons social Europeu Inverteix en el teu futur

An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa; Quorum sensing; VirulencePseudomonas aeruginosa; Detecció de quòrum; VirulènciaPseudomonas aeruginosa; Detección de quórum; VirulenciaUnderstanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections. IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment.This work has been funded by the Ministry of Science and Innovation (SAF2015-67476-R and RTI2018-096278-B-C21) and Fundación Marató de TV3 (TV32018-201825-30-31). The Nb4D group is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient: 2017 SGR 1441). CIBER-BBN is an initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation from 2013 to 2016, Iniciativa Ingenio 2010, Consolider Program, and CIBER Actions were financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Enrique J. Montagut and Juan Raya wish to thank the FPI fellowship (BES-2016-076496 and PRE2019-087542, respectively) from the Spanish Ministry of Science and Innovation. The Custom Antibody Service (CAbS) is acknowledged for its assistance and support in the production of HQNO antibodies

A new BiofilmChip device for testing biofilm formation and antibiotic susceptibility

Biofilms; Microbiologia clínica; Diagnòstic de malalties infecciosesBiofilms; Microbiología clínica; Diagnóstico de enfermedades infecciosasBiofilms; Clinical microbiology; Infectious-disease diagnosticsCurrently, three major circumstances threaten the management of bacterial infections: increasing antimicrobial resistance, expansion of chronic biofilm-associated infections, and lack of an appropriate approach to treat them. To date, the development of accelerated drug susceptibility testing of biofilms and of new antibiofouling systems has not been achieved despite the availability of different methodologies. There is a need for easy-to-use methods of testing the antibiotic susceptibility of bacteria that form biofilms and for screening new possible antibiofilm strategies. Herein, we present a microfluidic platform with an integrated interdigitated sensor (BiofilmChip). This new device allows an irreversible and homogeneous attachment of bacterial cells of clinical origin, even directly from clinical specimens, and the biofilms grown can be monitored by confocal microscopy or electrical impedance spectroscopy. The device proved to be suitable to study polymicrobial communities, as well as to measure the effect of antimicrobials on biofilms without introducing disturbances due to manipulation, thus better mimicking real-life clinical situations. Our results demonstrate that BiofilmChip is a straightforward tool for antimicrobial biofilm susceptibility testing that could be easily implemented in routine clinical laboratories.We thank Prof. Tim Tolker-Nielsen from Costerton Biofilm Center, University of Copenhagen, for providing the P. aeruginosa MK171 strain. This work was supported in part through grants to ET from the Ministerio de Ciencia, Innovación y Universidades (BIO2015-63557-R and RTI2018-098573-B-100) (MINECO/FEDER), the Generalitat de Catalunya (2017 SGR1079 and CERCA program), and the Spanish Cystic Fibrosis Foundation and La Caixa Foundation. The authors want to acknowledge MicroFabSpace and the Microscopy Characterization Facility, Unit 7 of ICTS “NANBIOSIS” from CIBER-BBN at IBEC. This research was supported by the Networking Biomedical Research Center (CIBER), Spain. CIBER is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III (RD16/0006/0012), with the support of the European Regional Development Fund. This work was funded by the CERCA Programme and by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2017 SGR 1079). This work was developed in the context of AdvanceCat and Base3D with the support of ACCIÓ (Catalonia Trade and Investment; Generalitat de Catalunya) under the Catalonian ERDF operational program (European Regional Development Fund) 2014–2020. This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) through the projects MINDS (Proyectos I+D Excelencia + FEDER): TEC2015-70104-P, CTQ2016-75870-P

An additional set of phages to characterize epidemic methicillin-resistant Staphylococcus aureus strains from Spain (1989-92).

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) isolates in Spain have increased dramatically; in 1986 there were only 1.2% MRSA amongst all nosocomial Staphylococcus aureus (SA) isolates, by 1989 this percentage had risen to 44% in some hospital causing a very serious epidemic situation in the country. We have characterized these isolates by direct, reverse and Fisk phage typing and we have also looked for an additional local set of phages to help us to differentiate these strains. We have been able to differentiate an epidemic strain from other MRSA strains which cause sporadic hospital outbreaks, and we have also distinguished between some variants of the epidemic strain.This research has been supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS), Ministerio de Sanidad y Consumo, Spain; No. 93/0144.S

Criptococosis pulmonar que simula un cáncer de pulmón

Cryptococcosis; Pulmonary malignancyCriptococcosi; Càncer de pulmóCriptococosis; Cáncer de pulmó

Global Emergence of Resistance to Fluconazole and Voriconazole in Candida parapsilosis in Tertiary Hospitals in Spain During the COVID-19 Pandemic

Candida parapsilosis; Antifungal resistance; OutbreaksCandida parapsilosis; Resistencia antifúngica; BrotesCandida parapsilosis; Resistència antifúngica; BrotsBackground Candida parapsilosis is a frequent cause of candidemia worldwide. Its incidence is associated with the use of medical implants, such as central venous catheters or parenteral nutrition. This species has reduced susceptibility to echinocandins, and it is susceptible to polyenes and azoles. Multiple outbreaks caused by fluconazole-nonsusceptible strains have been reported recently. A similar trend has been observed among the C. parapsilosis isolates received in the last 2 years at the Spanish Mycology Reference Laboratory. Methods Yeast were identified by molecular biology, and antifungal susceptibility testing was performed using the European Committee on Antimicrobial Susceptibility Testing protocol. The ERG11 gene was sequenced to identify resistance mechanisms, and strain typing was carried out by microsatellite analysis. Results We examined the susceptibility profile of 1315 C. parapsilosis isolates available at our reference laboratory between 2000 and 2021, noticing an increase in the number of isolates with acquired resistance to fluconazole, and voriconazole has increased in at least 8 different Spanish hospitals in 2020–2021. From 121 recorded clones, 3 were identified as the most prevalent in Spain (clone 10 in Catalonia and clone 96 in Castilla-Leon and Madrid, whereas clone 67 was found in 2 geographically unrelated regions, Cantabria and the Balearic Islands). Conclusions Our data suggest that concurrently with the coronavirus disease 2019 pandemic, a selection of fluconazole-resistant C. parapsilosis isolates has occurred in Spain, and the expansion of specific clones has been noted across centers. Further research is needed to determine the factors that underlie the successful expansion of these clones and their potential genetic relatedness.O.Z. was funded by grants SAF2017–86912-R and PID2020–114546RB-I00 from the Spanish Ministry for Science and Innovation. This work was also funded by the National Centre for Microbiology (Instituto de Salud Carlos III) through the Surveillance Program of Antifungal Resistance and the Center for Biomedical Research in Network of Infectious Diseases CIBERINFECTCB21/13/00105 (O.Z. and L.A.F.), CIBERINFEC-CB21/13/00009 (M.P.-A.), CIBERES-CB06/06/0037 (C.A.-T.), and CIBERES-CB06/06/0058 (J.G). L.A.-F. was supported by Fondo de Investigación Sanitaria (MPY 117/18 and MPY 305/20). We thank Dr. David Campany Herrero (Vall d’Hebron Hospital), Noelia Garrido Peño (Móstoles Hospital), David Gómez Gómez y Aitziber Illaro Uranga (Marqués de Valdecilla Hospital), María Ángeles Machín Morón (Burgos Hospital), Jose Manuel Caro Teller (Doce de Octubre Hospital), Marina Calvo (Puerta de Hierro Hospital), and Ariadna Padulles (Bellvitge Hospital) for providing the data on antifungal consumption from their hospitals. We also thank Ángel Zaballos and Pilar Jiménez from the Genomics Core Facility from Instituto de Salud Carlos III for their technical help with the microsatellite analysis technique

Influenza-Associated Disseminated Aspergillosis in a 9-Year-Old Girl Requiring ECMO Support

Nens; Influenza humana; IsavuconazolNiños; Influenza humana; IsavuconazolChildren; Human influenza; IsavuconazoleA previously healthy 9-year-old girl developed fulminant myocarditis due to severe influenza A infection complicated with methicillin-resistant Staphylococcus aureus pneumonia, requiring extracorporeal membrane oxygenation (ECMO) support. Twelve days after admission, Aspergillus fumigatus was isolated in tracheal aspirate, and 12 h later she suddenly developed anisocoria. Computed tomography (CT) of the head showed fungal brain lesions. Urgent decompressive craniectomy with lesion drainage was performed; histopathology found hyphae in surgical samples, culture-positive for Aspergillus fumigatus (susceptible to azoles, echinocandins, and amphotericin B). Extension workup showed disseminated aspergillosis. After multiple surgeries and combined antifungal therapy (isavuconazole plus liposomal amphotericin B), her clinical course was favorable. Isavuconazole therapeutic drug monitoring was performed weekly. Extensive immunological study ruled out primary immunodeficiencies. Fluorine-18 fluorodeoxyglucose positron emission tomography/CT (18F-FDG PET/CT) follow-up showed a gradual decrease in fungal lesions. Influenza-associated pulmonary aspergillosis is well-recognized in critically ill adult patients, but pediatric data are scant. Clinical features described in adults concur with those of our case. Isavuconazole, an off-label drug in children, was chosen because our patient had severe renal failure. To conclude, influenza-associated pulmonary aspergillosis is uncommon in children admitted to intensive care for severe influenza, but pediatricians should be highly aware of this condition to enable prompt diagnosis and treatment.This work received no external funding

Voriconazole Use in Children: Therapeutic Drug Monitoring and Control of Inflammation as Key Points for Optimal Treatment

Infeccions fúngiques pediàtriques; Monitorització terapèutica de fàrmacs; VoriconazolInfecciones fúngicas pediátricas; Monitorización terapéutica de fármacos; VoriconazolPaediatric fungal infections; Therapeutic drug monitoring; VoriconazoleVoriconazole plasma concentrations (PC) are highly variable, particularly in children. Dose recommendations in 2–12-year-old patients changed in 2012. Little data on therapeutic drug monitoring (TDM) after these new recommendations are available. We aimed to evaluate voriconazole monitoring in children with invasive fungal infection (IFI) after implementation of new dosages and its relationship with safety and effectiveness. A prospective, observational study, including children aged 2–12 years, was conducted. TDM was performed weekly and doses were changed according to an in-house protocol. Effectiveness, adverse events, and factors influencing PC were analysed. A total of 229 PC from 28 IFI episodes were obtained. New dosing led to a higher rate of adequate PC compared to previous studies; still, 35.8% were outside the therapeutic range. In patients aged < 8 years, doses to achieve therapeutic levels were higher than recommended. Severe hypoalbuminemia and markedly elevated C-reactive protein were related to inadequate PC. Therapeutic PC were associated with drug effectiveness and safety. Higher doses in younger patients and a dose adjustment protocol based on TDM should be considered. Voriconazole PC variability has decreased with current updated recommendations, but it remains high and is influenced by inflammatory status. Additional efforts to control inflammation in children with IFI should be encouraged.This research was funded by an Investigator Sponsored Research Grant from Pfizer (Grant Number WI182544