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Invasive fungal diseases are associated with significant morbidity and mortality, particularly in immunocompromised individuals. Early and accurate diagnosis is crucial for effective treatment. Despite traditional methods such as microbiological culture, histopathology, radiology and direct microscopy are available, antigen/antibody-based diagnostics are emerging for diagnosis of invasive fungal infections (IFI). Fungal cell wall is a unique structure composed of polysaccharides that are well correlated with fungal burden during fungal infections. Based on this feature, cell wall polysaccharides have been explored as antigens in IFIs diagnostics such as the galactomannan assay, mannan test, β-glucan assay and cryptococcal CrAg test. Herein, we provide an overview on the cell wall polysaccharides from three opportunistic pathogens: Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans, and their applications for IFIs diagnosis. The clinical outcome of newly developed cell wall polysaccharides-based diagnostics is also discussed., (© 2020 Blackwell Verlag GmbH.)

Detection of serum galactomannan (GM) and (1,3)-β-d-glucan (BG) is considered useful for non-culture diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. Only few studies evaluated these seromarkers in non-neutropenic patients suspected of having IPA. The aim of this study was to evaluate both tests together with the Aspergillus fumigatus-specific serum IgG and IgA (IgAG) test for serological IPA diagnosis in non-neutropenic patients. Sera from 87 patients suspected of having IPA were retrospectively analysed. Patients were categorised into groups of proven IPA (n = 10), putative IPA (n = 31) and non-IPA colonisation (n = 46). When the GM, BG and IgAG assays were used for patients included in the study, the sensitivity/specificity/positive predictive value (PPV)/negative predictive value (NPV) were 48.8%/91.3%/83.3%/66.7%, 82.9%/73.9%/73.9%/82.9% and 75.6%/95.7%/93.9%/81.5%, respectively. Thus, the highest specificity and PPV were confirmed for the IgAG assay. Improvements in the sensitivity and NPV were achieved by "at least one positive" analysis with the GM and BG assays, with the sensitivity/specificity/PPV/NPV values being 85.0%/69.6%/71.4%/84.2%. Nevertheless, the highest sensitivity and NPV were achieved by the "at least one positive" analysis combining the GM, BG and IgAG tests (97.6% and 96.8%, respectively). The involvement of the IgAG assay could improve IPA diagnosis in non-neutropenic patients by increasing the sensitivity and NPV when combined with the GM or BG assays. Furthermore, improvement was achieved by combining the GM, BG and IgAG assays using the "at least one positive test" strategy, especially if doubt exists., (© 2018 Blackwell Verlag GmbH.)

Aspergillus fumigatus is currently the major airborne fungal pathogen that menaces immunocompromised individuals. Germination of inhaled conidia is a hallmark of the early infection process, but little is known about the underlying mechanisms. The intention of our ongoing studies is the identification of A. fumigatus proteins that are differentially expressed during germination and may provide insights in the germination process. Using a proteomic approach, we identified AFUA_5G09330 as a major hyphal-specific protein. This result was confirmed using monoclonal antibodies generated in this study. AFUA_5G09330 belongs to a fungal-specific protein family. The eponymous CipC protein of A. nidulans has been shown to be induced by concanamycin A, and transcriptional data from Cryptococcus neoformans demonstrate a strong up-regulation of the expression of a homologous gene during infection. Our data provide evidence that AFUA_5G09330 is a monomeric, cytoplasmic protein. We found no evidence for an overexpression of AFUA_5G09330 induced by concanamycin A or other stress conditions. AFUA_5G09330 is exclusively found in the hyphal morphotype that enables an invasive growth of A. fumigatus during infection. Further studies are required to define the biological function of this hyphae-specific protein and its potential relevance for the pathogenicity of A. fumigatus.

A dihydropyridine derivative, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate (2e), having potent antifungal activity against pathogenic species of Aspergillus was investigated for its possible molecular mechanism of action. The SDS-PAGE coupled with nano-high-performance liquid chromatography-tandem mass spectrometry was used directly to assess both absolute abundance and differential expression of proteins in the secretory phases of Aspergillus fumigatus under the influence of 2e. It was observed that the compound inhibited the expression of two proteins of 60.99 and 79.77 kDa. Both of these secretory proteins that were inhibited by 2e, were analysed further by matrix assisted laser desorption ionization time-of-flight mass spectrometry. The 60.99- and 79.77-kDa proteins were identified as probable retroelement pol polyprotein and elongation factor G respectively. These targeted proteins could be the products of potentially virulence-related genes of A. fumigatus which may unravel the mode of action of 2e and pathobiology of A. fumigatus.

The incidence of life-threatening mycoses caused by opportunistic fungi has increased dramatically in recent years with members of the genera Candida and Aspergillus being the most commonly encountered species. Prompt initiation of antifungal therapy for good prognosis of such cases is highly dependent on accurate diagnosis. The potential of metabolic antigens in the diagnosis of aspergillosis was investigated in the present study. Two proteins of 18 and 70 kDa were identified with success rate of 35% and 60% respectively based on their reactivity with patient sera of clinically diagnosed cases of aspergillosis. The antibodies raised against 70 and 18 kDa proteins in rabbits were found to be useful in detection of A. fumigatus in the kidneys of a mouse model of aspergillosis.

Gliotoxin is produced by the fungus Aspergillus fumigatus. Aspergillus is widespread in the environment and this ubiquitous nature results in disease and co-carcinogenesis to be distributed world-wide. Gliotoxin contains an epipolythiodioxopiperazine (ETP) ring that is believed to be involved in redox reactions. The reactive oxygen species produced interact with DNA to form hydroxylated and other altered DNA products. To measure DNA adduct formation, we used 32P radiolabelling and, after enzymatic DNA digestion, separated adducts in two dimensions using thin-layer chromatography (2D-TLC), with ultimate autoradiography and densitometry. HeLa DNA was incubated with 0.1 mmol l-1 and 0.3 mmol l-1 of gliotoxin (and necessary redox agents) for 1 and 20 h. We found an increase in 6-hydro-5,6-dihydroxythymidine (thymine glycol) monophosphate [d(TG)MP] from 0.0% to 30.4%, an increase in 8-hydroxy-2'-deoxyguanidine monophosphate [8(OH)dGMP] from 0.0% to 4.2%, an increase in deoxynucleotide diphosphate (dNDP) from zero adducts to six DNA adducts, as well as an increase of other as yet unidentified adducts. Also, time exposure may have a greater effect than concentration based on a 20-h incubation with 0.3 mmol l-1 gliotoxin that completely obliterates the pyrimidines deoxythymidine 3'-monophosphate (dTMP) and deoxycytidine 3'-monophosphate (dCMP).

Forty-four isolates of Aspergillus fumigatus from patients with nosocomial aspergillosis and their hospital environment were collected and typed by immunoblot. Laser densitometric analysis of the profiles and homology studies within each case were performed which established relationships between patient and environmental types and showed that patients are often contaminated by several types of A. fumigatus, not all necessarily responsible for the mycosis. One of the cases appeared to be a typical clonal hospital-acquired infection. This typing technique seems to represent a powerful tool to determine the origin and mode of spread of nosocomial aspergillosis.