Detection of Specific IgE against Molds Involved in Allergic Bronchopulmonary Mycoses in Patients with Cystic Fibrosis.

CONTEXT: Allergic bronchopulmonary mycoses (ABPM) can be due to molds other than Aspergillus fumigatus in patients with cystic fibrosis (pwCF). We aimed to develop immunoassays for the detection of specific IgE (sIgE) directed against five fungal species involved in ABPM: Aspergillus terreus, Scedosporium apiospermum, Lomentospora prolificans, Rasamsonia argillacea, and Exophiala dermatitidis. MATERIALS AND METHODS: Serum samples (n = 356) from 238 pwCF, collected in eight CF care centers in France, Germany, and Italy, were analyzed by dissociated enhanced lanthanide fluorescent immunoassay (DELFIA®) to assess levels of sIgE directed against antigenic extracts of each fungus. Clinical, biological, and radiological data were collected for each episode. One hundred serum samples from healthy blood donors were used as controls. Sera were classified into four groups depending on the level of sIgE according to the quartile repartition calculated for the pwCF population. A score of 4 for values above the 3rd quartile corresponds to an elevated level of sIgE. RESULTS: PwCF showed higher levels of sIgE than controls. Based on criteria from the ABPA-ISHAM working group, with an additional criterion of "a sIgE score of 4 for at least one non-A. fumigatus mold", we were able to diagnose six cases of ABPM. CONCLUSIONS: Using 417 IU/mL as the threshold for total IgE and the same additional criterion, we identified seven additional pwCF with "putative ABPM". Detection of sIgE by DELFIA® showed good analytical performance and supports the role played by non-A. fumigatus molds in ABPM. However, commercially available kits usable in routine practice are needed to improve the diagnosis of ABPM.

Azole resistance in Aspergillus flavus and associated fitness cost.

BACKGROUND: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.

Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme.

Most bacterial, plant and fungal cells possess at their surface a protective layer called the cell wall, conferring strength, plasticity and rigidity to withstand the osmotic pressure. This molecular barrier is crucial for pathogenic microorganisms, as it protects the cell from the local environment and often constitutes the first structural component encountered in the host-pathogen interaction. In pathogenic molds and yeasts, the cell wall constitutes the main target for the development of clinically-relevant antifungal drugs. In the past decade, solid-state NMR has emerged as a powerful analytical technique to investigate the molecular organization of microbial cell walls in the context of intact cells. 13C NMR chemical shift is an exquisite source of information to identify the polysaccharides present in the cell wall, and two-dimensional 13C-13C correlation experiments provide an efficient tool to rapidly access the polysaccharide composition in whole cells. Here we investigate the use of the adiabatic DREAM (for dipolar recoupling enhancement through amplitude modulation) recoupling scheme to improve solid-state NMR analysis of polysaccharides in intact cells. We demonstrate the advantages of two-dimensional 13C-13C experiments using the DREAM recoupling scheme. We report the spectral editing of polysaccharide signals by varying the radio-frequency carrier position. We provide practical considerations for the implementation of DREAM experiments to characterize polysaccharides in whole cells. We demonstrate the approach on intact fungal cells of Neurospora crassa and Aspergillus fumigatus, a model and a pathogenic filamentous fungus, respectively. The approach could be envisioned to efficiently reduce the spectral crowding of more complex cell surfaces, such as cell wall and peptidoglycan in bacteria.

Deciphering Unexpected Vascular Locations of Scedosporium spp. and Lomentospora prolificans Fungal Infections, France.

Scedosporium spp. and Lomentospora prolificans are emerging non-Aspergillus filamentous fungi. The Scedosporiosis/lomentosporiosis Observational Study we previously conducted reported frequent fungal vascular involvement, including aortitis and peripheral arteritis. For this article, we reviewed 7 cases of Scedosporium spp. and L. prolificans arteritis from the Scedosporiosis/lomentosporiosis Observational Study and 13 cases from published literature. Underlying immunosuppression was reported in 70% (14/20) of case-patients, mainly those who had solid organ transplants (10/14). Osteoarticular localization of infection was observed in 50% (10/20) of cases; infections were frequently (7/10) contiguous with vascular infection sites. Scedosporium spp./Lomentospora prolificans infections were diagnosed in 9 of 20 patients ≈3 months after completing treatment for nonvascular scedosporiosis/lomentosporiosis. Aneurysms were found in 8/11 aortitis and 6/10 peripheral arteritis cases. Invasive fungal disease--related deaths were high (12/18 [67%]). The vascular tropism of Scedosporium spp. and L. prolificans indicates vascular imaging, such as computed tomography angiography, is needed to manage infections, especially for osteoarticular locations.

Lower airway microbiota compositions differ between influenza, COVID-19 and bacteria-related acute respiratory distress syndromes.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is responsible for 400,000 deaths annually worldwide. Few improvements have been made despite five decades of research, partially because ARDS is a highly heterogeneous syndrome including various types of aetiologies. Lower airway microbiota is involved in chronic inflammatory diseases and recent data suggest that it could also play a role in ARDS. Nevertheless, whether the lower airway microbiota composition varies between the aetiologies of ARDS remain unknown. The aim of this study is to compare lower airway microbiota composition between ARDS aetiologies, i.e. pulmonary ARDS due to influenza, SARS-CoV-2 or bacterial infection. METHODS: Consecutive ARDS patients according to Berlin's classification requiring invasive ventilation with PCR-confirmed influenza or SARS-CoV-2 infections and bacterial infections (> 105 CFU/mL on endotracheal aspirate) were included. Endotracheal aspirate was collected at admission, V3-V4 and ITS2 regions amplified by PCR, deep-sequencing performed on MiSeq sequencer (Illumina®) and data analysed using DADA2 pipeline. RESULTS: Fifty-three patients were included, 24 COVID-19, 18 influenza, and 11 bacterial CAP-related ARDS. The lower airway bacteriobiota and mycobiota compositions (ß-diversity) were dissimilar between the three groups (p = 0.05 and p = 0.01, respectively). The bacterial α-diversity was significantly lower in the bacterial CAP-related ARDS group compared to the COVID-19 ARDS group (p = 0.04). In contrast, influenza-related ARDS patients had higher lung mycobiota α-diversity than the COVID-19-related ARDS (p = 0 < 01). CONCLUSION: Composition of lower airway microbiota (both microbiota and mycobiota) differs between influenza, COVID-19 and bacterial CAP-related ARDS. Future studies investigating the role of lung microbiota in ARDS pathophysiology should take aetiology into account.

Detección y genotipificación de Pneumocystis jirovecii en muestras de pacientes mexicanos VIH positivos y negativos / Detection and genotyping of Pneumocystis jirovecii in samples from HIV positive and negative Mexican patients

Resumen Introducción: Pneumocystis jirovecii es un hongo atípico detectado particularmente en pacientes VIH-positivos o con trasplante. Objetivo: Detectar y genotipificar Pneumocystis jirovecii en muestras de pacientes de dos hospitales de la ciudad de México. Método: Fueron procesadas 89 muestras respiratorias, correspondientes a 53 pacientes (30 VIH positivos y 23 VIH negativos) con sintomatología respiratoria y 11 personas sanas incluidas como control negativo. El DNA fue extraído y amplificado por PCR anidada de la región del espaciador transcrito interno, obteniendo un fragmento en cada ronda (de 693 y 550 pb). Los genotipos y su relación filogenética fueron determinados por secuenciación del fragmento de 550 pb. Resultados: Cuarenta y ocho muestras de 30 pacientes VIH-positivos provenían de un solo hospital, de las cuales 11 (36.6 %) fueron positivas a Pneumocystis jirovecii. Ninguna fue positiva en pacientes VIH-negativos o personas sanas. Los haplotipos detectados con mayor frecuencia fueron Eg y Em. Conclusiones: La frecuencia de infección por Pneumocystis jirovecii fue alta en la población mexicana estudiada. El genotipo más frecuente fue diferente a los reportados en otros países. Es necesario encauzar este problema de salud hacia la detección temprana de esta infección.

Abstract Introduction: Pneumocystis jirovecii is an atypical fungus particularly detected in HIV-positive or transplant patients. Objective: To detect and genotype Pneumocystis jirovecii in patient samples from two hospitals in Mexico City. Method: Eighty-nine respiratory tract samples, corresponding to 53 patients (30 HIV-positive and 23 HIV-negative) with respiratory symptoms and to 11 healthy individuals included as negative control, were processed. DNA was extracted from the ITS region and amplified by nested polymerase chain reaction from the internal transcribed spacer, with one fragment being obtained at each round (693 and 550 bp). Genotypes and their phylogenetic relationship were determined by sequencing the 550 bp fragment. Results: Forty-eight samples from 30 HIV-positive patients were received from a single hospital, out of which 11 (36.6 %) were positive for Pneumocystis jirovecii. No sample was positive in HIV-negative patients or healthy subjects. The most frequently detected haplotypes were Eg and Em. Conclusions: The frequency of Pneumocystis jirovecii infection was high in the studied Mexican population. The most common genotype was different from those reported in other countries. It is necessary to address this health problem through early detection of this infection.

Aerially transmitted human fungal pathogens: what can we learn from metagenomics and comparative genomics? / Hongos de transmisión aérea patógenos para el ser humano: conocimientos adquiridos a partir de su metagenómica y genómica comparativa

In the last few decades, aerially transmitted human fungal pathogens have been increasingly recognized to impact the clinical course of chronic pulmonary diseases, such as asthma, cystic fibrosis or chronic obstructive pulmonary disease. Thanks to recent development of culture-free high-throughput sequencing methods, the metagenomic approaches are now appropriate to detect, identify and even quantify prokaryotic or eukaryotic microorganism communities inhabiting human respiratory tract and to access the complexity of even low-burden microbe communities that are likely to play a role in chronic pulmonary diseases. In this review, we explore how metagenomics and comparative genomics studies can alleviate fungal culture bottlenecks, improve our knowledge about fungal biology, lift the veil on cross-talks between host lung and fungal microbiota, and gain insights into the pathogenic impact of these aerially transmitted fungi that affect human beings. We reviewed metagenomic studies and comparative genomic analyses of carefully chosen microorganisms, and confirmed the usefulness of such approaches to better delineate biology and pathogenesis of aerially transmitted human fungal pathogens. Efforts to generate and efficiently analyze the enormous amount of data produced by such novel approaches have to be pursued, and will potentially provide the patients suffering from chronic pulmonary diseases with a better management. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

En las últimas décadas se ha reconocido cada vez más la influencia de los hongos patógenos para el ser humano, y cuya transmisión es aérea, en el curso clínico de afecciones pulmonares crónicas, como el asma, la fibrosis quística o la enfermedad pulmonar obstructiva crónica. Gracias al desarrollo reciente de métodos de secuenciación de alto rendimiento, que no requieren cultivo, en la actualidad los análisis metagenómicos permiten detectar, identificar e incluso cuantificar comunidades de microorganismos procariotas o eucariotas que habitan en las vías respiratorias del ser humano, y acceder a la complejidad de las comunidades microbianas cuya población es de baja densidad, que posiblemente desempeñan un papel en las enfermedades pulmonares crónicas. En la presente revisión examinamos cómo los estudios metagenómicos y genómicos comparativos pueden ayudar a superar los obstáculos de los cultivos de hongos, mejorar nuestros conocimientos sobre la biología fúngica, desvelar el diálogo cruzado (crosstalk) entre el pulmón del huésped y la microbiota fúngica asociada, y adquirir información sobre la influencia patogénica de estos hongos transmitidos por el aire que afectan al ser humano. Revisamos los estudios metagenómicos y los análisis genómicos comparativos de microorganismos cuidadosamente seleccionados, y confirmamos la utilidad de estas estrategias para definir mejor la biología y la patogenia de hongos de transmisión aérea que son patógenos para el ser humano. Los esfuerzos por generar y analizar eficientemente la ingente cantidad de datos obtenidos con estos nuevos métodos deberán continuar, y es posible que ofrezcan un mejor tratamiento de los pacientes portadores de enfermedades pulmonares crónicas.Este manuscrito forma parte de la serie de artículos presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Population structure and diversity of the pathogenic fungus Aspergillus fumigatus isolated from different sources and geographic origins

Fifty-five clinical and environmental Aspergillus fumigatus isolates from Mexico, Argentina, France and Peru were analyzed to determine their genetic variability, reproductive system and level of differentiation using amplified fragment length polymorphism markers. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective alleles, expected heterozygocity and by performing an association index test (I A). The degree of genetic differentiation and variation was determined using analysis of molecular variance at three levels. Using the paired genetic distances, a dendrogram was built to detect the genetic relationship among alleles. Finally, a network of haplotypes was constructed to determine the geographic relationship among them. The results indicate that the clinical isolates have greater genetic variability than the environmental isolates. The I A of the clinical and environmental isolates suggests a recombining population structure. The genetic differentiation among isolates and the dendrogram suggest that the groups of isolates are different. The network of haplotypes demonstrates that the majority of the isolates are grouped according to geographic origin.