Filamentous fungi present significant health hazards to immunocompromised individuals globally; however, the prompt and precise identification of them during infection remains challenging. In this study, a TaqMan probe-based multiplex real-time PCR (M-qPCR) assay was developed to detect simultaneously the target genes of four important pathogenic filamentous fungi: ANXC4 gene of Aspergillus fumigatus, EF1-α gene of Fusarium spp., mitochondrial rnl gene of Mucorales, and hcp100 gene of Histoplasma capsulatum. In this M-qPCR assay, the limit of detection (LoD) to all four kinds of fungi was 100 copies and the correlation coefficients (R2) were above 0.99. The specificity of this assay is 100%, and the minimum detection limit is 100 copies/reaction. In conclusion, an M-qPCR detection assay was well established with high specificity and sensitivity for rapid and simultaneous detection on four important filamentous fungi in the clinic.
OBJECTIVE: World Health Organization developed the first fungal priority pathogens list (WHO FPPL) in 2022. Aspergillus fumigatus, Mucorales, Fusarium spp., and Histoplasma spp. are the four types of pathogenic fungi with filamentous morphology in the critical priority group and high priority group of WHO FPPL. These four filamentous fungal infections have become more common and severe in immunocompromised patients with the increase in susceptible populations in recent decades, which resulted in a substantial burden on the public health system. However, prompt and precise identification of them during infection remains challenging. Our study established successfully a TaqMan probe-based multiplex real-time qPCR assay for four clinically important filamentous fungi, A. fumigatus, Fusarium spp., Mucorales, and Histoplasma capsulatum, with high sensitivity and specificity, which shows promising potential for prompt and precise diagnosis against fungal infection.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

During investigations of invertebrate-associated fungi in Yunnan Province of China, a new species, Sporodiniella sinensis sp. nov., was collected. Morphologically, S. sinensis is similar to Sporodiniella umbellata; however, it is distinguished from S. umbellata by its greater number of sporangiophore branches, longer sporangiophores, larger sporangiospores, and columellae. The novel species exhibits similarities of 91.62 % for internal transcribed spacer (ITS), 98.66-99.10 % for ribosomal small subunit (nrSSU), and 96.36-98.22 % for ribosomal large subunit (nrLSU) sequences, respectively, compared to S. umbellata. Furthermore, phylogenetic analyses based on combined sequences of ITS, nrLSU and nrSSU show that it forms a separate clade in Sporodiniella, and clusters closely with S. umbellata with high statistical support. The phylogenetic and morphological evidence support S. sinensis as a distinct species. Here, it is formally described and illustrated, and compared with other relatives.

The influence of four carbon and nitrogen substrates on the quality and flavor of a novel surimi-based product fermented with Actinomucor elegans (A. elegans) was investigated, with a focus on carbon and nitrogen catabolite repression. The results showed that the substrate significantly affected mycelial growth, enzyme activities, and the metabolites of A. elegans. Although glucose significantly promoted A. elegans growth by 116.69%, it decreased enzyme secretion by 69.79% for α-amylase and 59.80% for protease, most likely by triggering the carbon catabolite repression pathway. Starch, soy protein, and wheat gluten substantially affected the textural properties of the fermented surimi. Furthermore, wheat gluten significantly promoted the protease activity (102.70%) and increased protein degradation during surimi fermentation. The fishy odor of surimi was alleviated through fermentation, and a correlation between the volatile compounds and A. elegans metabolism was observed. These results explore fermentation substrates in filamentous fungi metabolism from a catabolite repression perspective.

CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA .

OBJECTIVE: Given the high fatality rates, prompt and accurate identification of the fungal culprit is crucial, emphasizing the need for invasive mucormycosis. Unfortunately, mucormycosis lacks definitive biomarkers, depending primarily on smears, cultures, or pathology, all necessitating invasive specimen collection from the infection site. However, obtaining valid specimens early in critically ill patients poses substantial risks and challenges. Whether peripheral blood metagenomic next-generation sequencing (mNGS) can enhance early mucormycosis diagnosis, especially when direct specimen collection from the infection site is challenging, is warranted. This is a large-scale clinical study conducted to evaluate the utility and clinical impact of mNGS of peripheral blood for the diagnosis of invasive mucormycosis. We believe our study provided both novelty in translational medicine and a great value for the medical community to understand the strengths and limitations of mNGS of peripheral blood as a new diagnostic tool for the diagnosis and management of invasive mucormycosis.

The species within the family Cunninghamellaceae are widely distributed and produce important metabolites. Morphological studies along with a molecular phylogeny based on the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA revealed two new species in this family from soils in China, that is, Absidia ovalispora sp. nov. and Cunninghamella globospora sp. nov. The former is phylogenetically closely related to Absidia koreana, but morphologically differs in sporangiospores, sporangia, sporangiophores, columellae, collars, and rhizoids. The latter is phylogenetically closely related to Cunninghamella intermedia, but morphologically differs in sporangiola and colonies. They were described and illustrated.

OBJECTIVE: Since systematic antifungals for mucormycosis showed variable MICs depending on strains, effective and safe antifungal therapy was still needed. This study is aimed to evaluate the in vitro activity of doxycycline combined with antifungal therapy against dominant Mucorales pathogens.
METHODS: Multidrug susceptibility testing was performed with doxycycline and antifungals, including itraconazole, posaconazole, and amphotericin, in 21 isolates of 8 dominant Mucorales pathogens.
RESULTS: The fractional inhibitory concentration index according to M38 showed one Rhizopus arrhizus isolate synergic (∑FICI = 0.375) and other isolates in addition (0.5 < ∑FICI < 4).
CONCLUSIONS: Doxycycline was found to have in vitro advantages in combined antifungal treatment over antifungals alone.

Mucoralean fungi could cause mucormycosis in humans, particularly in immunodeficient individuals and those with diabetes mellitus or trauma. With plenty of species and genera, their molecular identification and pathogenicity have a large deviation. Reported cases of mucormycosis showed frequent occurrence in Rhizopus species, Mucor species, and Lichtheimia species. We analyzed the whole genome sequences of 25 species of the top 10 Mucorales genera, along with another 22 important pathogenic non-Mucorales species, to dig the target genes for monitoring Mucorales species and identify potential genomic imprints of virulence in them. Mucorales-specific genes have been found in various orthogroups extracted by Python script, while genus-specific genes were annotated covering cellular structure, biochemistry metabolism, molecular processing, and signal transduction. Proteins related to the virulence of Mucorales species varied with distinct significance in copy numbers, in which Orthofinder was conducted. Based on our fresh retrospective analysis of mucormycosis, a comparative genomic analysis of pathogenic Mucorales was conducted in more frequent pathogens. Specific orthologs between Mucorales and non-Mucoralean pathogenic fungi were discussed in detail. Referring to the previously reported virulence proteins, we included more frequent pathogenic Mucorales and compared them in Mucorales species and non-Mucorales species. Besides, more samples are needed to further verify the potential target genes.

Rhino-orbital-cerebral mucormycosis (ROCM), which is an acute fatal infectious disease with a high mortality rate, is increasingly being diagnosed in patients with hematological diseases worldwide. We aimed to investigate the clinical characteristics, treatment, and prognosis of hematological diseases complicated by ROCM. Our sample comprised a total of 60 ROCM patients with hematological diseases. The most common primary disease was acute lymphoblastic leukemia (ALL) (n=27, 45.0%), while 36 patients (60.0%) were diagnosed with a clear type of pathogen, all belonging to the Mucorales, most commonly Rhizopus (41.7%). Of the 32 patients (53.3%) who died, 19 (59.3%) died of mucormycosis, and 84.2% (n=16) of those died within 1 month. Forty-eight cases (80.0%) received antifungal treatment combined with surgical therapy, 12 of whom (25.0%) died of mucormycosis, amounting to a mortality rate that was significantly lower than in patients who received antifungal therapy alone (n=7, 58.3%) (P=0.012). The median neutrophil value of patients who underwent surgery was 0.58 (0.11-2.80) 103/μL, the median platelet value was 58.00 (17.00-93.00) 103/μL, and no surgery-related deaths were reported. Multivariate analysis showed that patient\'s advanced age (P=0.012, OR=1.035 (1.008-1.064)) and lack of surgical treatment (P=0.030, OR=4.971 (1.173-21.074)) were independent prognostic factors.In this study, hematological diseases associated with ROCM have a high mortality rate. Lack of surgical treatment is an independent prognostic factor for death from mucormycosis. Surgery may therefore be considered in patients with hematological disease even if their neutrophil and platelet values are lower than normal.