High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

LGR5 as a Therapeutic Target of Antibody-Functionalized Biomimetic Magnetoliposomes for Colon Cancer Therapy.

Purpose: The lack of specificity of conventional chemotherapy is one of the main difficulties to be solved in cancer therapy. Biomimetic magnetoliposomes are successful chemotherapy controlled-release systems, hyperthermia, and active targeting agents by functionalization of their surface with monoclonal antibodies. The membrane receptor Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) stands out as colorectal cancer (CRC) biomarker and appears to be related to treatment resistance and the development of metastasis. The aim of this study was to assess the effectiveness and safety of LGR5-targeted biomimetic magnetoliposomes loaded with oxaliplatin (OXA) or 5-fluorouracil (5-FU) in the selective treatment of CRC and their possible application in hyperthermia. Methods: Synthesis, characterization and determination of heating capacity of magnetoliposomes transporting OXA or 5-FU (with and without LGR5 functionalization) were conducted. In vitro antitumoral activity was assayed in multiple colorectal cell lines at different times of exposition. In addition to this, cell internalization was studied by Prussian Blue staining, flow cytometry and fluorescence microscopy. In vivo acute toxicity of magnetoliposomes was performed to evaluate iron-related toxicity. Results: OXA and 5-FU loaded magnetoliposomes functionalized with LGR5 antibody showed higher cellular uptake than non-targeted nanoformulation with a reduction of the percentage of proliferation in colon cancer cell lines up to 3.2-fold of the IC50 value compared to that of free drug. The differences between non-targeted and targeted nanoformulations were more evident after short exposure times (4 and 8 hours). Interestingly, assays in the MC38 transduced cells with reduced LGR5 expression (MC38-L(-)), showed lower cell internalization of LGR5-targeted magnetoliposomes compared to non-transduced MC38 cell line. In addition, magnetoliposomes showed an in vitro favorable heating response under magnetic excitation and great iron-related biocompatibility data in vivo. Conclusion: Drug-loaded magnetoliposomes functionalized with anti-LGR5 antibodies could be a promising CRC treatment strategy for LGR5+ targeted chemotherapy, magnetic hyperthermia, and both in combination.

Factores asociados a la adherencia al tratamiento farmacológico en pacientes con diabetes sin hipertensión arterial

Objetivo: Identificar los factores asociados a la adherencia al tratamiento farmacológico en pacientes con diabetes mellitus tipo 2 sin hipertensión arterial. Materiales y métodos: Estudio transversal y analítico en pacientes con diabetes mellitus tipo 2 sin hipertensión arterial. Se integraron el grupo con adherencia al tratamiento (n = 145) y el grupo sin adherencia al tratamiento (n = 49), determinado por la escala de adherencia a la medicación de Morisky de 4 ítems (MMAS-4). Como factores asociados, se estudiaron sexo, vida en pareja, escolaridad, ocupación, edad agrupada en decenios y tiempo de evolución de la diabetes agrupada en menos de un año, de 1 a 5 años, de 6 a 10 años y más de 10 años. El plan de análisis estadístico incluyó la prueba de ji al cuadrado. Resultados: Se identificaron el sexo (p = 0,045), la vida en pareja (p = 0,045), la edad (p = 0,001) y el tiempo de evolución de la enfermedad (p = 0,001) como factores asociados a la adherencia al tratamiento farmacológico. La escolaridad no se identificó como un factor asociado a la adherencia terapéutica en el paciente con diabetes mellitus tipo 2 (p = 0,289). A mayor edad, menor adherencia al tratamiento farmacológico en el paciente con diabetes: el punto de corte se presenta a los 60 años; después de esta edad, el porcentaje en el grupo no adherente es mayor que en el grupo con adherencia (p = 0,001). A mayor tiempo de evolución de la diabetes, menor probabilidad de adherencia; en el grupo no adherente, el porcentaje de pacientes con más de 10 años de evolución es 67,3 % y en el grupo con adherencia el porcentaje corresponde a 33,8 % (p = 0,001). Conclusiones: Se identificaron los factores asociados a la adherencia al tratamiento farmacológico en pacientes con diabetes mellitus tipo 2 sin hipertensión arterial.

Objective: To identify the factors associated with adherence to drug therapy among patients with type 2 diabetes mellitus without hypertension. Materials and methods: A cross-sectional, analytical study conducted with patients with type 2 diabetes mellitus without hypertension, divided into an adherent group (n = 145) and a non-adherent group (n = 49), determined by the 4-item Morisky Medication Adherence Scale (MMAS-4). The associated factors were sex, cohabitation, schooling, occupation, age in 10-year groups and diabetes progression time grouped in less than 1 year, from 1 to 5 years, from 6 to 10 years and more than 10 years. The statistical analysis plan included the chi-square test. Results: The factors associated with adherence to drug therapy were identified as sex (p = 0.045), cohabitation (p = 0.045), age (p = 0.001) and disease progression time (p = 0.001). Schooling was not identified as a factor associated with adherence to drug therapy among patients with type 2 diabetes mellitus (p = 0.289). The older the patient with diabetes, the lower the adherence to drug therapy; the cut-off point was 60 years of age, after which the percentage in the non-adherent group was higher than in the adherent group (p = 0.001). The longer the diabetes progression time, the lower the probability of adherence; in the non-adherent group the percentage of patients with more than 10 years of disease progression was 67.3 % and in the adherent group the percentage was 33.8 % (p = 0.001). Conclusions: The factors associated with adherence to drug therapy among patients with type 2 diabetes mellitus without hypertension were identified

mAb-Functionalized Biomimetic MamC-Mediated-Magnetoliposomes as Drug Delivery Systems for Cancer Therapy.

In cancer therapy, new therapeutic nanoformulations able to mediate targeted chemotherapy are required. Recently, biomimetic magnetic nanoparticles (BMNPs) mediated by MamC, a magnetosome protein from Magnetococcus marinus MC-1, have proven, in vitro and in vivo, to be effective drug nanocarriers (following the application of an external gradient magnetic field) and to allow combination with hyperthermia. However, these nanoassemblies require further optimization to improve cytocompatibility, stability and active targeting ability. Herein, we describe the production of the magnetoliposomes (LP) embedding BMNPs functionalized (or not) with doxorubicin (DOXO), [LP(+/-DOXO-BMNPs)], and their surface modification with the DO-24 mAb, which targets the human Met/HGF receptor's ectodomain (overexpressed in many cancers). Nanoformulations were extensively characterized using TEM, DLS, FTIR and when tested in vitro, the lipid coating increased the colloidal stability and their biocompatibility, favoring the cellular uptake in cells overexpressing the cognate receptor. Indeed, the magnetoliposomes mAb-LP(+/-DOXO-BMNPs) exerted a specific active targeting ability by the presence of the mAb that preserved its immunocompetence. Both LP(BMNPs) and mAb-LP(BMNPs) were not toxic to cells, while +/-mAb-LP(DOXO-BMNPs) nanoformulations were indeed cytotoxic. Therefore, this study represents a proof of concept for the development of promising drug carriers for cancer therapy based on local chemotherapy directed by mAbs.

Magnetite Mineralization inside Cross-Linked Protein Crystals.

Crystallization in confined spaces is a widespread process in nature that also has important implications for the stability and durability of many man-made materials. It has been reported that confinement can alter essential crystallization events, such as nucleation and growth and, thus, have an impact on crystal size, polymorphism, morphology, and stability. Therefore, the study of nucleation in confined spaces can help us understand similar events that occur in nature, such as biomineralization, design new methods to control crystallization, and expand our knowledge in the field of crystallography. Although the fundamental interest is clear, basic models at the laboratory scale are scarce mainly due to the difficulty in obtaining well-defined confined spaces allowing a simultaneous study of the mineralization process outside and inside the cavities. Herein, we have studied magnetite precipitation in the channels of cross-linked protein crystals (CLPCs) with different channel pore sizes, as a model of crystallization in confined spaces. Our results show that nucleation of an Fe-rich phase occurs inside the protein channels in all cases, but, by a combination of chemical and physical effects, the channel diameter of CLPCs exerted a precise control on the size and stability of those Fe-rich nanoparticles. The small diameters of protein channels restrain the growth of metastable intermediates to around 2 nm and stabilize them over time. At larger pore diameters, recrystallization of the Fe-rich precursors into more stable phases was observed. This study highlights the impact that crystallization in confined spaces can have on the physicochemical properties of the resulting crystals and shows that CLPCs can be interesting substrates to study this process.

Nanoassemblies of acetylcholinesterase and ß-lactamase immobilized on magnetic nanoparticles as biosensors to detect pollutants in water.

The use of enzymes immobilized on magnetic nanoparticles to detect contaminants in aqueous samples has gained interest, since it allows the magnetic control, concentration and reuse of the enzymes. In this work, the detection of trace amounts of organophosphate pesticides (chlorpyrifos) and antibiotics (penicillin G) in water was attained by developing a nanoassembly formed by either inorganic or biomimetic magnetic nanoparticles used as substrates to immobilize acetylcholinesterase (AChE) and ß-lactamase (BL). Other than the substrate, the optimization of the nanoassembly was done by testing enzyme immobilization both through electrostatic interaction (also reinforced with glutaraldehyde) and covalent bonds (by carbodiimide chemistry). Temperature (25 °C), ionic strength (150 mM NaCl) and pH (7) were set to ensure enzymatic stability and to allow both the nanoparticles and the enzymes to present ionic charges that would allow electrostatic interaction. Under these conditions, the enzyme load on the nanoparticles was ⁓0.1 mg enzyme per mg nanoparticles, and the preserved activity after immobilization was 50-60% of the specific activity of the free enzyme, being covalent bonding the one which yielded better results. Covalent nanoassemblies could detect trace concentrations of pollutants down to 1.43 nM chlorpyrifos and 0.28 nM penicillin G. They even permitted the quantification of 14.3 µM chlorpyrifos and 2.8 µM penicillin G. Also, immobilization conferred higher stability to AChE (⁓94% activity after 20 days storage at 4 °C) and allowed to reuse the BL up to 12 cycles.

Influence of the Nature of Aminoalcohol on ZnO Films Formed by Sol-Gel Methods.

Here we present comparative studies of: (i) the formation of ZnO thin films via the sol-gel method using zinc acetate dihydrate (ZAD), 2-methoxyethanol (ME) as solvent, and the aminoalcohols (AA): ethanolamine, (S)-(+)-2-amino-1-propanol, (S)-(+)-2-amino-3-methyl-1-butanol, 2-aminophenol, and aminobenzyl alcohol, and (ii) elemental analyses, infrared spectroscopy, X-ray diffraction, scanning electron microscopy, absorption and emission spectra of films obtained after deposition by drop coating on glass surface, and thermal treatments at 300, 400, 500 and 600 °C. The results obtained provide conclusive evidences of the influence of the AA used (aliphatic vs. aromatic) on the ink stability (prior to deposition), and on the composition, structures, morphologies, and properties of films after calcination, in particular, those due to the different substituents, H, Me, or iPr, and to the presence or the absence of a -CH2 unit. Aliphatic films, more stable and purer than aromatic ones, contained the ZnO wurtzite form for all annealing temperatures, while the cubic sphalerite (zinc-blende) form was also detected after using aromatic AAs. Films having frayed fibers or quartered layers or uniform yarns evolved to "neuron-like" patterns. UV and photoluminescence studies revealed that these AAs also affect the optical band gap, the structural defects, and photo-optical properties of the films.

Effects of Magnetic Nanoparticles on the Functional Activity of Human Monocytes and Dendritic Cells.

The use of nanoparticles in medicine is sometimes hampered by their potential to activate immune cells, eliciting inflammation or allergy. We investigated whether magnetic nanoparticles (MNPs) or biomimetic magnetic nanoparticles (BMNPs) affect relevant activities of human monocytes. We found that the nanoparticles neither elicited the production of pro-inflammatory mediators IL-6 and TNFα by resting monocytes (when BMNP dose < 300 µg/mL) nor enhanced their secretion induced by R848, a molecule engaging virus-recognizing receptors, or bacterial lipopolysaccharide (LPS). MNPs and BMNPs neither induced the generation of reactive oxygen species (ROS), nor affected the ROS production elicited by the NADPH oxidase activator phorbol myristate acetate (PMA) or the fungal derivative ß-glucan. BMNPs, but not MNPs, caused an up-regulation of the maturation markers CD80, CD83, and CD86 in immature monocyte-derived dendritic cells (DCs), whereas both nanoparticles did not affect the LPS-induced expression of these markers. Moreover, the nanoparticles were greedily ingested by monocytes and DCs without altering their viability. Therefore, these nanoparticles are candidates for medical applications because they do not activate pro-inflammatory activities of monocytes. Furthermore, their ability to stimulate DC maturation could be used for the design of vaccines. Moreover, harmlessly engulfed nanoparticles could be vehicles to carry molecules inside the immune cells to regulate the immune response.

Effectiveness of the BNT162b2 mRNA Covid-19 vaccine in Spanish healthcare workers.

INTRODUCTION: Frontline health care workers (HCW) have higher risk than the general population to become infected by SARS.CoV.2, so they were a priority group for Covid-19 vaccine administration. We compared the incidence and prevalence of HCW infected pre-and post-vaccination with the BNT162b2 mRNA COVID-19 vaccine. MATERIAL AND METHODS: Prospective observational study carried out between 01/12/20 and 07/03/21 in La Paz University Hospital, Madrid (Spain). SARS.CoV.2 positive cases in HCW after vaccination were collected and compared to those hospitalized COVID-19 patients at the same hospital. RESULTS: Two weeks after finishing the first round of vaccinations daily new cases of HCW infections (symptomatic and asymptomatic) decreased substantially and cumulative cases of infected HCW and hospitalized COVID-19 patients started to diverge. No new positive cases of HCW infection were registered seven days after the second dose of BNT162b2 mRNA COVID-19 vaccine. CONCLUSIONS: BNT162b2 mRNA COVID-19 vaccine is highly effective in Spanish HCW.

Effectiveness of the BNT162b2 mRNA Covid-19 vaccine in Spanish healthcare workers.

Introduction: Frontline health care workers (HCW) have higher risk than the general population to become infected by SARS.CoV.2, so they were a priority group for Covid-19 vaccine administration. We compared the incidence and prevalence of HCW infected pre-and post-vaccination with the BNT162b2 mRNA COVID-19 vaccine. Material and methods: Prospective observational study carried out between 01/12/20 and 07/03/21 in La Paz University Hospital, Madrid (Spain). SARS.CoV.2 positive cases in HCW after vaccination were collected and compared to those hospitalized COVID-19 patients at the same hospital. Results: Two weeks after finishing the first round of vaccinations daily new cases of HCW infections (symptomatic and asymptomatic) decreased substantially and cumulative cases of infected HCW and hospitalized COVID-19 patients started to diverge. No new positive cases of HCW infection were registered seven days after the second dose of BNT162b2 mRNA COVID-19 vaccine. Conclusions: BNT162b2 mRNA COVID-19 vaccine is highly effective in Spanish HCW.

Introducción: Los trabajadores sanitarios (TS) de primera línea tienen mayor riesgo de infectarse de SARS-CoV-2 que la población general, por lo que han sido un grupo prioritario para la vacunación frente a COVID-19. Comparamos la incidencia y prevalencia de TS infectados antes y después de la vacunación con BNT162b2 mRNA frente a COVID-19. Material y métodos: Estudio prospectivo observacional realizado entre 01 de diciembre de 2021 en el Hospital Universitario La Paz, Madrid, España. Se registraron los casos positivos para SARS-CoV-2 en TS y se compararon con los hospitalizados por COVID-19. Resultados: Dos semanas tras la primera ronda de vacunación las nuevas infecciones en TS (sintomáticas y asintomáticos) disminuyeron sustancialmente y los casos acumulados de TS infectados y pacientes hospitalizados por COVID-19 empezaron a divergir. No hubo nuevas infecciones en TS vacunados a los siete días de la segunda dosis de la vacuna. Conclusión: La vacuna BNT162b2 mRNA frente a SARS-CoV-2 es altamente eficaz en TS españoles.

Embedding Biomimetic Magnetic Nanoparticles Coupled with Peptide AS-48 into PLGA to Treat Intracellular Pathogens.

Among the strategies employed to overcome the development of multidrug-resistant bacteria, directed chemotherapy combined with local therapies (e.g., magnetic hyperthermia) has gained great interest. A nano-assembly coupling the antimicrobial peptide AS-48 to biomimetic magnetic nanoparticles (AS-48-BMNPs) was demonstrated to have potent bactericidal effects on both Gram-positive and Gram-negative bacteria when the antimicrobial activity of the peptide was combined with magnetic hyperthermia. Nevertheless, intracellular pathogens remain challenging due to the difficulty of the drug reaching the bacterium. Thus, improving the cellular uptake of the nanocarrier is crucial for the success of the treatment. In the present study, we demonstrate the embedding cellular uptake of the original nano-assembly into THP-1, reducing the toxicity of AS-48 toward healthy THP-1 cells. We optimized the design of PLGA[AS-48-BMNPs] in terms of size, colloidal stability, and hyperthermia activity (either magnetic or photothermal). The stability of the nano-formulation at physiological pH values was evaluated by studying the AS-48 release at this pH value. The influence of pH and hyperthermia on the AS-48 release from the nano-formulation was also studied. These results show a slower AS-48 release from PLGA[AS-48-BMNPs] compared to previous nano-formulations, which could make this new nano-formulation suitable for longer extended treatments of intracellular pathogens. PLGA[AS-48-BMNPs] are internalized in THP-1 cells where AS-48 is liberated slowly, which may be useful to treat diseases and prevent infection caused by intracellular pathogens. The treatment will be more efficient combined with hyperthermia or photothermia.

Antimicrobial defenses of table eggs: Importance of antibacterial proteins in egg white as a function of hen age in an extended production cycle.

The importance of egg natural defences to prevent bacterial contamination and their relation with hen age in extended production cycles were evaluated. Egg-white from eggs of different hen age groups (up 100-weeks-old) and lines (Hy-Line white and brown) were inoculated with Gram-positive Staphylococcus aureus or Gram-negative Salmonella Typhimurium, ranging from 103-106 CFU/mL. Our results show that concentrations of egg-white lysozyme and, particularly, ovotransferrin are important to modulate bacterial survival in a dose-dependent matter. Depending on protein concentration, their effect ranges from bactericidal to bacteriostatic, with a threshold for bacterial contamination that depends also on hen age and line. The concentrations of lysozyme and ovotransferrin increased with hen age (up to 2 and 22 w/w% of total protein, respectively), and eggs laid by older hens exhibited the greatest potential to prevent the growth of the highest Salmonella inoculum (106 CFU/mL). Salmonella-penetration experiments demonstrated that non-contaminated eggs display significantly higher concentrations of antimicrobial proteins. However, eggs from older hens needed a higher concentration of these proteins (>20% ovotransferrin) to prevent bacterial contamination, showing that antimicrobial protein concentrations in egg-whites was not the only factor influencing bacterial contamination. Finally, this study demonstrated that egg-white of eggs produced by old hens are less prone to contamination by Salmonella.

Biogeochemical fingerprinting of magnetotactic bacterial magnetite.

Biominerals are important archives of the presence of life and environmental processes in the geological record. However, ascribing a clear biogenic nature to minerals with nanometer-sized dimensions has proven challenging. Identifying hallmark features of biologically controlled mineralization is particularly important for the case of magnetite crystals, resembling those produced by magnetotactic bacteria (MTB), which have been used as evidence of early prokaryotic life on Earth and in meteorites. We show here that magnetite produced by MTB displays a clear coupled C-N signal that is absent in abiogenic and/or biomimetic (protein-mediated) nanometer-sized magnetite. We attribute the presence of this signal to intracrystalline organic components associated with proteins involved in magnetosome formation by MTB. These results demonstrate that we can assign a biogenic origin to nanometer-sized magnetite crystals, and potentially other biominerals of similar dimensions, using unique geochemical signatures directly measured at the nanoscale. This finding is significant for searching for the earliest presence of life in the Earth's geological record and prokaryotic life on other planets.

Combination of Photodynamic Therapy and Oral Antifungals for the Treatment of Onychomycosis.

Onychomycosis accounts for 50% of nail disorders, making it one of the most prevalent fungal diseases and a therapeutic challenge. Photodynamic therapy (PDT) could constitute a therapeutic alternative, owing to its good adherence, the low probability of resistance, the lack of interaction with antimicrobials, and its favorable adverse effect profile. This retrospective observational study included all patients with a microbiological diagnosis of onychomycosis treated with PDT at Miguel Servet University Hospital, Zaragoza (Spain), between January 2013 and June 2021. The protocol consisted of pre-treatment with 40% urea for 7 days, followed by 16% methyl-aminolevulinate (MAL) for 3 h and subsequent irradiation with a red-light LED lamp (37 J/cm2), every 1 or 2 weeks. Combined treatment with oral and/or topical antifungals was recorded. Of the 20 patients included (mean age, 59 ± 17 years), 55% were men. The most frequently detected microorganism was Trichophyton rubrum (55%). The most commonly affected location was the feet (90%): 50% of these cases were associated with tinea pedis. The median (standard deviation) number of PDT sessions was 6 (2.8). PDT was combined with systemic terbinafine (250 mg/day) in 10 cases (in 8 cases, this was administered for only 1 month), and with topical terbinafine in 3 cases. A complete clinical response was achieved in 80% (16) of cases and microbiological cure in 60% (12). PDT is a therapeutic alternative for onychomycosis, and can be administered either in monotherapy or combined with antifungals, allowing for a reduction in the duration and possible adverse effects of antifungal treatment and achieving higher cure rates than those obtained with either treatment alone.

Successful control of Serratia marcescens outbreak in a neonatal unit of a tertiary-care hospital in Spain.

OBJECTIVE: Serratia marcescens is a Gram-negative bacterium that is found in hospital environments and commonly associated with outbreaks in neonatal units. One S. marcescens isolate was detected from a bloodstream culture from a neonate in our hospital that was followed by an outbreak. The aim of this study was to describe the molecular epidemiology of a S. marcescens outbreak in the neonatal unit. METHODS: In order to investigate the outbreak, weekly surveillance rectal swabs were submitted for culture from all patients admitted in this unit from August to September 2018. Environmental samples were obtained from potential sources in September 2018. Typing of isolates was performed by pulsed field gel electrophoresis (PFGE). In addition, we studied the in vitro activity of chlorhexidine against S. marcescens. RESULTS: During this period, 146 infants were hospitalised in our neonatal unit, of which 16 patients had a S. marcescens-positive sample. A total of 36 environmental surveillance samples were collected, and one sample from a stethoscope from an incubator of a colonized baby was positive for S. marcescens. All the 18 isolates, including the isolate from the stethoscope, belonged to a single PFGE cluster. We found that very low concentrations of chlorhexidine, even with application times close to 0 achieved significant reductions in the amount of S. marcescens. CONCLUSION: A unique clone of S. marcescens caused this outbreak, including isolates from patients and from one stethoscope. The outbreak was controlled with the early implementation of specific control measures.

Integration of In Silico and In Vitro Analysis of Gliotoxin Production Reveals a Narrow Range of Producing Fungal Species.

Gliotoxin is a fungal secondary metabolite with impact on health and agriculture since it might act as virulence factor and contaminate human and animal food. Homologous gliotoxin (GT) gene clusters are spread across a number of fungal species although if they produce GT or other related epipolythiodioxopiperazines (ETPs) remains obscure. Using bioinformatic tools, we have identified homologous gli gene clusters similar to the A. fumigatus GT gene cluster in several fungal species. In silico study led to in vitro confirmation of GT and Bisdethiobis(methylthio)gliotoxin (bmGT) production in fungal strain cultures by HPLC detection. Despite we selected most similar homologous gli gene cluster in 20 different species, GT and bmGT were only detected in section Fumigati species and in a Trichoderma virens Q strain. Our results suggest that in silico gli homology analyses in different fungal strains to predict GT production might be only informative when accompanied by analysis about mycotoxin production in cell cultures.

Detection of azole resistance in Aspergillus fumigatus complex isolates using MALDI-TOF mass spectrometry.

OBJECTIVES: The main goal of this study was to accurately detect azole resistance in species of the Aspergillus fumigatus complex by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). METHODS: Identification of isolates (n = 868) was done with MALDI-TOF MS using both commercial and in-house libraries. To determine azole susceptibility, the EUCAST E.Def. 9.3.2 method was applied as the reference standard. Identification of resistant isolates was confirmed by DNA sequence analysis. Protein spectra obtained by MALDI-TOF MS were analysed to differentiate species within the A. fumigatus complex and to detect azole-resistant A. fumigatus sensu stricto isolates. RESULTS: Correct discrimination of A. fumigatus sensu stricto from cryptic species was accomplished in 100% of the cases applying principal component analysis (PCA) to protein spectra generated by MALDI-TOF MS. Furthermore, a specific peak (4586 m/z) was found to be present only in cryptic species. The application of partial least squares (PLS) discriminant analysis allowed 98.43% (±0.038) discrimination between susceptible and azole-resistant A. fumigatus sensu stricto isolates. Finally, based on PLS and SVM, A. fumigatus sensu stricto isolates with different cyp51A gene mutations were correctly clustered in 91.5% of the cases. CONCLUSIONS: MALDI-TOF MS combined with peak analysis is a novel tool that allows the differentiation of A. fumigatus sensu stricto from other species within the A. fumigatus complex, as well as the detection of azole-resistant A. fumigatus sensu stricto. Although further studies are still needed, the results reported here show the great potential of MALDI-TOF and machine learning for the rapid detection of azole-resistant Aspergillus fumigatus isolates from clinical origins.