Evaluation of metagenomic next-generation sequencing (mNGS) combined with quantitative PCR: cutting-edge methods for rapid diagnosis of non-invasive fungal rhinosinusitis.

PURPOSE: Fungal rhinosinusitis is a significant and growing health concern in arid regions, with an increasing incidence over recent decades. Without timely and appropriate management, it can lead to severe complications, including potential intracranial spread. This study aims to establish efficient and rapid diagnostics for non-invasive fungal rhinosinusitis (FRS), addressing the challenge of its difficult-to-culture diagnosis. METHODS: Twenty-eight patients suspected of FRS were studied using endoscopic sinus surgery to obtain tissue samples for histopathology, direct microscopy, fungal culture, quantitative PCR (qPCR) and metagenomic next-generation sequencing (mNGS) detection. A patented qPCR targeting prevalent Aspergillus species was evaluated. RESULTS: The patient cohort had a male-to-female ratio of 9:14, with disease duration up to 50 years. Histopathologically, 23 out of 28 cases were positive. Fungal culture exhibited a sensitivity of 21.74%, with one false positive. qPCR and mNGS showed 100% sensitivity and specificity, with a 100% consistency rate for identification at the species level (23/23), and potential detection of cases with co-infections. The most common pathogen was A. flavus, followed by A. fumigatus and A. niger. Two cases involved mixed infections of A. fumigatus and A. flavus. CONCLUSION: qPCR and mNGS proved effective in rapidly identifying fungi from fresh sinus tissue that are challenging to culture, surpassing conventional methods. However, further evaluation and optimization with a larger cohort of patients are necessary. Histopathology is still recommended to confirm the clinical significance of the detected fungal species.

Aflatoxin profiles of Aspergillus flavus isolates in Sudanese fungal rhinosinusitis.

Aspergillus flavus is a commonly encountered pathogen responsible for fungal rhinosinusitis (FRS) in arid regions. The species is known to produce aflatoxins, posing a significant risk to human health. This study aimed to investigate the aflatoxin profiles of A. flavus isolates causing FRS in Sudan. A total of 93 clinical and 34 environmental A. flavus isolates were studied. Aflatoxin profiles were evaluated by phenotypic (thin-layer and high-performance chromatography) and genotypic methods at various temperatures and substrates. Gene expression of aflD and aflR was also analyzed. A total of 42/93 (45%) isolates were positive for aflatoxin B1 and AFB2 by HPLC. When the incubation temperature changed from 28°C to 36°C, the number of positive isolates decreased to 41% (38/93). Genetic analysis revealed that 85% (79/93) of clinical isolates possessed all seven aflatoxin biosynthesis-associated genes, while 27% (14/51) of non-producing isolates lacked specific genes (aflD/aflR/aflS). Mutations were observed in aflS and aflR genes across both aflatoxin-producers and non-producers. Gene expression of aflD and aflR showed the highest expression between the 4th and 6th days of incubation on the Sabouraud medium and on the 9th day of incubation on the RPMI (Roswell Park Memorial Institute) medium. Aspergillus flavus clinical isolates demonstrated aflatoxigenic capabilities, influenced by incubation temperature and substrate. Dynamic aflD and aflR gene expression patterns over time enriched our understanding of aflatoxin production regulation. The overall findings underscored the health risks of Sudanese patients infected by this species, emphasizing the importance of monitoring aflatoxin exposure.

Aspergillus flavus, mainly causing fungal rhinosinusitis in Sudan, poses health risks due to aflatoxin production. This study revealed diverse levels of aflatoxin and gene expression of clinical isolates by pheno- and genotypic methods, emphasizing the need for vigilant monitoring in the region.

Fungal primary and opportunistic pathogens: an ecological perspective.

Fungal primary pathogenicity on vertebrates is here described as a deliberate strategy where the host plays a role in increasing the species' fitness. Opportunism is defined as the coincidental survival of an individual strain in host tissue using properties that are designed for life in an entirely different habitat. In that case, the host's infection control is largely based on innate immunity, and the etiologic agent is not transmitted after infection, and thus fungal evolution is not possible. Primary pathogens encompass two types, depending on their mode of transmission. Environmental pathogens have a double life cycle, and tend to become enzootic, adapted to a preferred host in a particular habitat. In contrast, pathogens that have a host-to-host transmission pattern are prone to shift to a neighboring, immunologically naive host, potentially leading to epidemics. Beyond these prototypical life cycles, some environmental fungi are able to make large leaps between dissimilar hosts/habitats, probably due to the similarity of key factors enabling survival in an entirely different niche, and thus allowing a change from opportunistic to primary pathogenicity. Mostly, such factors seem to be associated with extremotolerance.

Difference analysis of the structure and functional properties among coix seed prolamin fractions (α-, ß-, and γ-coixin).

The differences in proteins in structural characteristics are reported to affect their physicochemical and functional properties. In this study, three types of prolamins (γ-, α-, and ß-coixin) derived from coix seed separately distributed among fractions 1-3 extracts. They were studied respecting molecular weight, amino acid composition, secondary structure, microstructure, surface hydrophobicity, solubility, water holding capacity, and oil holding capacity. Results showed that the molecular weights of those three fractions were between 10 and 40 kDa. The secondary structure of those fractions was almost the same, mainly based on ß-sheet and irregular structure. The microstructure of α- and γ-coixin presented an irregular shape, whereas ß-coixin presented a regular spherical shape. Those three fractions exhibited species of abundant essential amino acids with the same amino acid composition but different contents. The ß-coixin fraction had the highest content of hydrophobic amino acids (238.39 mg/g) followed by the α-coixin fraction (235.05 mg/g), whereas the γ-coixin fraction had the lowest content (33.27 mg/g). The γ-coixin fraction has the maximum surface hydrophobicity, whereas the ß-coixin fraction has the highest solubility. In addition, the good amphiphilicity of ß-coixin fraction made it possible to be used as a surfactant. The excellent functional properties of the ß-coixin fraction presented in this research would widen the applications of coix seed prolamins. PRACTICAL APPLICATION: The molecular weights of those three fractions were between 10 and 40 kDa. The secondary structure was almost the same, mainly based on ß-sheet and irregular structure. Those three fractions exhibited species of abundant essential amino acids with the same amino acid composition but different contents. The WHC and OHC of ß-coixin were the best, indicating its potential as a surfactant and forming stable lotion.

Cultivar difference characterization of kiwifruit wines on phenolic profiles, volatiles and antioxidant activity.

Antioxidant activity and volatiles of kiwifruit wine with different flesh colors were investigated in this study. Green (Guichang and Xuxiang), red (Donghong and Hongyang), and yellow (Jinyan) kiwifruits were analyzed to determine their alcohol content, phenolic profiles, antioxidant activity, and aroma composition. The results showed that Hongyang and Donghong wines had higher antioxidant activity and content of antioxidant substances. Hongyang wine possessed the most abundance of polyphenolic compounds, chlorogenic acid and catechins were the main polyphenols of kiwi wines. The 101 aromatic components were detected, Xuxiang wine possessed 64 aromatic compounds, Donghong and Hongyang wines had the higher esters compositions, 79.87%, and 78.0% respectively. From PCA (Principal Component Analysis), the volatile substances of kiwi wine with the same flesh color were similar. Five kinds of kiwi wines shared 32 kinds of volatile compounds, these compounds may be the core volatiles in kiwi wine. Therefore, the color of kiwi flesh can impact wine flavor, with Hongyang and Donghong kiwis owning red flesh being the most suitable for producing kiwi wine which would be a new milestone to the wine manufactures.

Fungi involved in rhinosinusitis in arid regions: insights from molecular identification and antifungal susceptibility.

Fungal rhinosinusitis (FRS) is a common problem worldwide, with an increasing burden in arid climate regions. Aspergillus species are the most common causative agents involved. In the present study, we investigated the prevalence, molecular characterization, and antifungal susceptibility of opportunists causing FRS in Sudan on the basis of strains collected over a period of 5 years. ß-Tubulin and calmodulin sequencing were used for species identification, and antifungal susceptibility profiles were evaluated by the protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Predominant species belonged to the Aspergillus flavus complex (n = 244), A. terreus complex (n = 16), A. fumigatus complex (n = 7), and other fungi (n = 17). Molecular identification of 94 strains of Aspergillus revealed the following species: A. flavus (n = 88), A. terreus (n = 1), A. citrinoterreus (n = 2), A. fumigatus (n = 1), A. caespitosus (n = 1), and A. sydowii (n = 1). Several A. flavus and an A. fumigatus isolates showed reduced susceptibility to azoles (minimum inhibitory concentrations above the clinical breakpoints or epidemiological cutoff values). Despite several mutations revealed in cyp51A of these isolates, none could be directly linked to azole resistance. Molecular identification of fungi causing FRS is useful to identify cryptic species and for epidemiologic studies. IMPORTANCE Fungal rhinosinusitis (FRS) is a significant clinical problem in arid regions. This study provides new insights into the prevalence, etiology, and antifungal susceptibility of FRS pathogens in Sudan, where the disease burden is high. Aspergillus species, particularly the A. flavus complex, were identified as the primary FRS pathogens in the region, with some evidence of antifungal resistance. The molecular identification of fungal species causing FRS is useful for detecting antifungal resistance, identifying cryptic species, and characterizing the epidemiology of the disease. The emergence of Azole resistance Aspergilli in Sudan highlights the need for continued surveillance and appropriate use of antifungal agents. These findings have important implications for clinical management, public health policy, and future research on FRS. Publishing this study in Microbiology Spectrum would enable other researchers and clinicians to build on these findings, ultimately improving the diagnosis, treatment, and prevention of FRS.

[Experimental study on the potential carcinogenicity of cooking oil fume condensate].

The potential carcinogenicity of cooking oil fume condensate (COFC) to human was studied. Human embryo lung diploid fibroblast cell strain KMB-17 cell was applied to establish a human diploid cell transformation system in vitro. Different concentrations of COFC were added into the media and co-incubated with cells. The malignant degree of transformation was assessed by the biological characteristics of the cells. The concentrations of COFC within the dose range of the experiment could induce the malignant transformation of KMB-17 cell, and with a obvious dose-response relationship (r = 0.9811). Transformed cells have exhibited many characteristics associating with malignant transformation, such as loss of density and contact-dependent inhibition, growth at low serum concentration, agglutination by low concentration of Con A, alteration of karyotype from diploid to aneuploid, and lose of anchorage dependence. It suggested that the malignant transformation of human embryo lung diploid fibroblast cell strain KMB-17 cell could be induced by COFC, which might have potential carcinogenicity to human.