Introduction
Quantitative reverse transcription PCR (RT‐qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Given that it will almost certainly continue ...to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits.
Methods and results
In this study, combinations of four primers/probe sets were used to construct a flexible RT‐qPCR assay which is capable of discriminating between SARS‐CoV‐2 and the seasonal human coronavirus HCoV‐OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed.
Conclusions
This study provides an efficient method for the simultaneous detection of SARS‐CoV‐2, HCoV‐OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results.
Significance and impact of the study
The multiplex RT‐qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.
Lomentospora (Scedosporium) prolificans is an opportunistic pathogen capable of causing invasive infections in immunocompromised patients. The fungus is able to disseminate via the bloodstream ...finally arriving at the central nervous system producing neurological symptoms and, in many cases, patient death. In this context, microglial cells, which are the resident immune cells in the central nervous system, may play an important role in these infections. However, this aspect of anti‐L. prolificans immunity has been poorly researched to date. Thus, the interactions and activity of microglial cells against L. prolificans were analysed, and the results show that there was a remarkable impairment in their performance regarding phagocytosis, the development of oxidative burst, and in the production of pro‐inflammatory cytokines, compared with macrophages. Interestingly, L. prolificans displays great growth also when challenged with immune cells, even when inside them. We also proved that microglial phagocytosis of the fungus is highly dependent on mannose receptor and especially on dectin‐1. Taken together, these data provide evidence for an impaired microglial response against L. prolificans and contribute to understanding the pathobiology of its neurotropism.
The filamentous fungus Lomentospora (Scedosporium) prolificans is an emerging opportunistic pathogen associated with fatal infections in patients with disturbed immune function. Unfortunately, ...conventional therapies are hardly of any use against this fungus due to its intrinsic resistance. Therefore, we performed an integrated study of the L. prolificans responses to the first option to treat these mycoses, namely voriconazole, with the aim of unveiling mechanisms involved in the resistance to this compound. To do that, we used a wide range of techniques, including fluorescence and electron microscopy to study morphological alterations, ion chromatography to measure changes in cell-wall carbohydrate composition, and proteomics-based techniques to identify the proteins differentially expressed under the presence of the drug. Significantly, we showed drastic changes occurring in cell shape after voriconazole exposure, L. prolificans hyphae being shorter and wider than under control conditions. Interestingly, we proved that the architecture and carbohydrate composition of the cell wall had been modified in the presence of the drug. Specifically, L. prolificans constructed a more complex organelle with a higher presence of glucans and mannans. In addition to this, we identified several differentially expressed proteins, including Srp1 and heat shock protein 70 (Hsp70), as the most overexpressed under voriconazole-induced stress conditions. The mechanisms described in this study, which may be directly related to L. prolificans antifungal resistance or tolerance, could be used as targets to improve existing therapies or to develop new ones in order to successfully eliminate these mycoses.
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•Quantification of fumagillin produced by different strains of A. fumigatus.•First validated method applied to the quantification of fumagillin in cell culture.•Fast, simple and ...sensitive determination of fumagillin by SPE-UHPLC-DAD.•Useful method to study invasive aspergillosis.
Fumagillin is a biomolecule produced by Aspergillus fumigatus that is gaining relevance due to its connection with invasive aspergillosis. The determination of this molecule might help to understand the propagation of this disease and study its use as a potential biomarker. In spite of the interest of fumagillin in microbiological research, no quantitative method has been developed so far for its determination in cell culture media. In this work, the first validated method for the quantitative analysis of fumagillin in RPMI-1640 is presented. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled the validation criteria established by EMA and FDA guidelines for bioanalysis (selectivity, carry over, linearity, accuracy, precision, dilution integrity and stability) and offers a limit of quantitation (25 μg·L−1) suitable for its intended use. Indeed, the method was satisfactorily applied to the quantification of the fumagillin produced by three strains of Aspergillus fumigatus with different toxin production capacity.
Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus
. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality ...rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the
fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by
, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology.
In inflammatory bowel diseases (IBDs), such as Crohn’s disease (CD) and ulcerative colitis (UC), the immune system relentlessly attacks intestinal cells, causing recurrent tissue damage over the ...lifetime of patients. The etiology of IBD is complex and multifactorial, involving environmental, microbiota, genetic, and immunological factors that alter the molecular basis of the organism. Among these, the microbiota and immune cells play pivotal roles; the microbiota generates antigens recognized by immune cells and antibodies, while autoantibodies target and attack the intestinal membrane, exacerbating inflammation and tissue damage. Given the altered molecular framework, the analysis of multiple molecular biomarkers in patients proves exceedingly valuable for diagnosing and prognosing IBD, including markers like C reactive protein and fecal calprotectin. Upon detection and classification of patients, specific treatments are administered, ranging from conventional drugs to new biological therapies, such as antibodies to neutralize inflammatory molecules like tumor necrosis factor (TNF) and integrin. This review delves into the molecular basis and targets, biomarkers, treatment options, monitoring techniques, and, ultimately, current challenges in IBD management.
Immune disorders arise from complex genetic and environmental factors, which lead to dysregulation at the cellular and inflammatory levels and cause tissue damage. Recent research highlights the ...crucial role of reactive antibodies in autoimmune diseases and graft rejection, but their complex determination poses challenges for clinical use. Therefore, our study aimed to ascertain whether the presence of reactive antibodies against membrane antigens in tissues from both animal models and humans could serve as biomarkers in patients with autoimmune disorders. To address this issue, we examined the binding profile of serological antibodies against a diverse panel of cell membranes from the spleen, liver, and kidney tissues of monkeys, rats, and humans. After developing the cell membrane microarrays, human sera were immunologically assayed. The study was first conducted on sera from two groups, healthy subjects and patients with inflammatory and autoimmune disorders, and then optimized for kidney transplant patient sera. A significant increase in antibody reactivity against specific monkey kidney and spleen membranes was observed in the serum of patients with lupus nephritis, while kidney transplant patients showed a significant enhancement against human tissues and human embryonic kidney 293 cells. These results show the potential importance for clinical and basic research purposes of studying the presence of specific IgG against membrane antigens in patients' serum as potential biomarkers of immune disorders. However, it is important to note that these results need to be verified in further studies with a larger sample size to confirm their relevance.
Purpose
The study of the immunocompetent airways immune response may provide important information to improve the therapeutic efficacy against Lomentospora (Scedosporium) prolificans. So, this study ...aimed to identify the most prevalent conidial antigens of this multiresistant fungus recognized by healthy human salivary immunoglobulin A, and to study their expression and cross‐reactivity with other fungal species.
Experimental design
Twenty saliva from immunocompetent donors were used to detect and identify the immunoreactive proteins by 2D immunoblotting and LC‐MS/MS. Moreover, anti‐Aspergillus antibodies were purified to study their cross‐reactivity.
Results
Ten proteins of L. prolificans conidia showed reactivity with more than 50% of the saliva samples. Among them, cyclophilin and enolase were the most prevalent antigens recognized by 85 and 80% of the samples, respectively. These enzymes were also identified on the cell wall surface of L. prolificans and on the immunomes of Scedosporium apiospermum and Scedosporium aurantiacum. Additionally, they showed cross‐reactivity with the most common pathogenic filamentous fungus Aspergillus fumigatus.
Conclusion and clinical relevance
These results show that the immunocompetent immune response might offer a pan‐fungal recognition of conserved antigens such as enolase and cyclophilins, making them potential candidates for study as therapeutic targets.
In this study, two distinct
infection models of
, using murine macrophages (RAW264.7) and human lung epithelial cells (A549), were employed to identify the genes important for fungal adaptation ...during infection. Transcriptomic analyses of co-incubated
uncovered 140 fungal genes up-regulated in common between both models that, when compared with a previously published
transcriptomic study, allowed the identification of 13 genes consistently up-regulated in all three infection conditions. Among them, the
gene, responsible for a critical step in the L-phenylalanine degradation pathway, was identified. Disruption of
resulted in a mutant strain unable to complete the Phe degradation pathway, leading to an excessive production of pyomelanin when this amino acid served as the sole carbon source. Moreover, the disruption mutant exhibited noticeable cell wall abnormalities, with reduced levels of β-glucans within the cell wall but did not show lack of chitin or mannans. The
mutant strain induced reduced inflammation in primary macrophages and displayed significantly lower virulence in a neutropenic mouse model of infection. This is the first study linking the
gene to fungal cell wall homeostasis and virulence.
The detection and diagnosis of the opportunistic fungi
spp. and
still relies mainly on low-sensitive culture-based methods. This fact is especially worrying in Cystic Fibrosis (CF) patients in whom ...these fungal species are frequently isolated and may increase the risk of suffering from an infection or other health problems. Therefore, with the purpose of developing a serologic detection method for
/
, four different
protein extracts (whole cell protein extract, secretome, total cell surface and conidial surface associated proteins) were studied by ELISA to select the most useful for IgG detection in sera from CF patients. The four extracts were able to discriminate the
/
-infected from
infected and non-infected patients. However, the whole cell protein extract was the one selected, as it was the one with the highest output in terms of protein concentration per ml of fungal culture used, and its discriminatory capacity was the best. The ELISA test developed was then assayed with 212 sera from CF patients and it showed to be able to detect
spp. and
with very high sensitivity and specificity, 86%-100% and 93%-99%, respectively, depending on the cut-off value chosen (four values were proposed A
= 0.5837, A
= 0.6042, A
= 0.6404, and A
= 0.7099). Thus, although more research is needed to reach a standardized method, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, allowing the monitoring of the humoral response to fungal presence.
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