BACKGROUND: Mucormycosis is an aggressive, invasive fungal infection caused by moulds in the order Mucorales. Early diagnosis is key to improving patient prognosis, yet relies on insensitive culture or non-specific histopathology. A pan-Mucorales specific monoclonal antibody (mAb), TG11, was recently developed. Here, we investigate the spatio-temporal localisation of the antigen and specificity of the mAb for immunohistochemistry.
METHODS: We use immunofluorescence (IF) microscopy to assess antigen localisation in eleven Mucorales species of clinical importance and live imaging of Rhizopus arrhizus germination. Immunogold transmission electron microscopy (immunoTEM) reveals the sub-cellular location of mAb TG11 binding. Finally, we perform immunohistochemistry of R. arrhizus in an ex vivo murine lung infection model alongside lung infection by Aspergillus fumigatus.
RESULTS: IF revealed TG11 antigen production at the emerging hyphal tip and along the length of growing hyphae in all Mucorales except Sakasenea. Timelapse imaging revealed early antigen exposure during spore germination and along the growing hypha. ImmunoTEM confirmed mAb TG11 binding to the hyphal cell wall only. The TG11 mAb specifically stained Mucorales but not Aspergillus hyphae in infected murine lung tissue.
CONCLUSIONS: TG11 detects early hyphal growth and has valuable potential for diagnosing mucormycosis by enhancing discriminatory detection of Mucorales in tissue.

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.

Sternal osteomyelitis and mediastinitis are rare yet severe complications post-cardiac surgery, often associated with significant morbidity and mortality. Fungal etiologies, particularly those caused by Rhizopus spp., are infrequent but can lead to aggressive infections. Here, we present the case of a 68-year-old male who developed sternal osteomyelitis and mediastinitis caused by Rhizopus spp. two weeks following coronary artery bypass grafting surgery. Debridement and pectoralis flap reconstruction were performed following clinical identification and confirmation with microbiological examinations and a CT scan. Prompt recognition, aggressive surgical intervention, and targeted antifungal therapy were crucial for successful management. This case underscores the importance of considering fungal pathogens, such as Rhizopus, in the differential diagnosis of post-cardiac surgery infections, as well as aggressive treatment to improve outcomes for affected patients.

UNASSIGNED: The mainstay of treatment for COVID-19-associated mucormycosis was liposomal Amphotericin B. Other antifungal agents, such as posaconazole and isavuconazole, were used as well. The Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing recommend broth microdilution methods for antifungal susceptibility testing. In this regard, the present study aimed to see what potency and zone diameters correlate with the gold standard broth microdilution method.
UNASSIGNED: All the isolates were identified by matrix-assisted laser desorption ionization-time-of-flight. In total, 127 isolates of 83 Rhizopus oryzae complex and 44 isolates of Rhizopus microsporus complex were selected. Anti-fungal susceptibility testing by disc diffusion and E-test was performed on Mueller Hinton Agar and compared with the CLSI broth microdilution method of Anti-fungal susceptibility testing.
UNASSIGNED: Percentage agreement was found to be more in the case of the E test than the disc diffusion method. In the case of R. oryzae, posaconazole had 98.79% agreement with broth microdilution followed by Isavuconazole (97.59%), Itraconazole (96.38%), and Amphotericin B (91.56%).
UNASSIGNED: Disc diffusion correlates well with broth microdilution, although its correlation is weaker when compared to the E test. Effective concentration of Amphotericin B discs for antifungal susceptibility testing depends on the specific Rhizopus species.

Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the Mucorales order. Among the Mucor species complex, Mucor circinelloides is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance. With this new model, phenotypic analyses of gene deletion mutants were conducted to define Erg3 and Erg6a as key biosynthetic enzymes in the M. circinelloides ergosterol pathway. Erg3 is a C-5 sterol desaturase involved in growth, sporulation, virulence, and azole susceptibility. In other fungal pathogens, erg3 mutations confer azole resistance because Erg3 catalyzes the production of a toxic diol upon azole exposure. Surprisingly, M. circinelloides produces only trace amounts of this toxic diol and yet, it is still susceptible to posaconazole and isavuconazole due to alterations in membrane sterol composition. These alterations are severely aggravated by erg3Δ mutations, resulting in ergosterol depletion and, consequently, hypersusceptibility to azoles. We also identified Erg6a as the main C-24 sterol methyltransferase, whose activity may be partially rescued by the paralogs Erg6b and Erg6c. Loss of Erg6a function diverts ergosterol synthesis to the production of cholesta-type sterols, resulting in resistance to amphotericin B. Our findings suggest that mutations or epimutations causing loss of Erg6 function may arise during human infections, resulting in antifungal drug resistance to first-line treatments against mucormycosis.
OBJECTIVE: The Mucor species complex comprises a variety of opportunistic pathogens known to cause mucormycosis, a potentially lethal fungal infection with limited therapeutic options. The only effective first-line treatments against mucormycosis consist of liposomal formulations of amphotericin B and the triazoles posaconazole and isavuconazole, all of which target components within the ergosterol biosynthetic pathway. This study uncovered M. circinelloides Erg3 and Erg6a as key enzymes to produce ergosterol, a vital constituent of fungal membranes. Absence of any of those enzymes leads to decreased ergosterol and consequently, resistance to ergosterol-binding polyenes such as amphotericin B. Particularly, losing Erg6a function poses a higher threat as the ergosterol pathway is channeled into alternative sterols similar to cholesterol, which maintain membrane permeability. As a result, erg6a mutants survive within the host and disseminate the infection, indicating that Erg6a deficiency may arise during human infections and confer resistance to the most effective treatment against mucormycoses.

Rhino-orbital-cerebral mucormycosis (ROCM) is linked to uncontrolled diabetes, diabetic ketoacidosis, iron overload, corticosteroid therapy, and neutropenia. This study evaluated a commercial real-time PCR system\'s effectiveness in detecting Mucorales from nasal swabs in 50 high-risk patients. Nasal swab PCR showed 30% positivity, compared to 8% with KOH microscopy. Despite its improved sensitivity, nasal swab PCR has limitations, highlighting the importance of established sampling methods in mucormycosis diagnosis. Participants were predominantly male (64%), with diabetes (78%) and amphotericin B use (96%). Prior COVID-19 was 42%, with 30% positive for Mucorales by PCR, compared to 8% with KOH microscopy.

The species within the family Cunninghamellaceae during an investigation of soil microfungi in Korea, in which three strains were isolated from Gangwon, Chungbuk, and Gyeongbuk provinces, designated as KNUF-22-121A, KNUF-22-126A, and KNUF-22-316, respectively. Because the morphological and molecular analyses of these three strains were identical, KNUF-22-316 underwent further detailed study. Phylogenetic analyses based on the concatenated nucleotide sequences of the internal transcribed spacer region and the large subunit 28S rRNA gene revealed that the strain belonged to the genus Absidia, but occupied a distinct phylogenetic position. The strain KNUF-22-316 was compared with closely related species Absidia radiata CGMCC 3.16257T and Absidia yunnanensis CGMCC 3.16259T, morphologically different with shorter sporangiophores, smaller sporangia and columellae, and the consistent presence of collars. Here, we provide a detailed description and images of this proposed new species, which we have named Absidia microsporangia sp. nov.

The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal priority pathogens list (FPPL). This systematic review aimed to evaluate the epidemiology and impact of invasive fungal disease due to Mucorales. PubMed and Web of Science were searched to identify studies published between January 1, 2011 and February 23, 2021. Studies reporting on mortality, inpatient care, complications and sequelae, antifungal susceptibility, risk factors, preventability, annual incidence, global distribution, and emergence during the study time frames were selected. Overall, 24 studies were included. Mortality rates of up to 80% were reported. Antifungal susceptibility varied across agents and species, with the minimum inhibitory concentrations lowest for amphotericin B and posaconazole. Diabetes mellitus was a common risk factor, detected in 65%-85% of patients with mucormycosis, particularly in those with rhino-orbital disease (86.9%). Break-through infection was detected in 13.6%-100% on azole or echinocandin antifungal prophylaxis. The reported prevalence rates were variable, with some studies reporting stable rates in the USA of 0.094-0.117/10 000 discharges between 2011 and 2014, whereas others reported an increase in Iran from 16.8% to 24% between 2011 and 2015. Carefully designed global surveillance studies, linking laboratory and clinical data, are required to develop clinical breakpoints to guide antifungal therapy and determine accurate estimates of complications and sequelae, annual incidence, trends, and global distribution. These data will provide robust estimates of disease burden to refine interventions and better inform future FPPL.

During surge of COVID-19-associated mucormycosis (CAM), we identified five cases of CAM where two different species of Mucorales were isolated. All had history of diabetes mellitus and presented with clinical features suggesting rhino-orbital mucormycosis. The patients grew different species from their nasal scraping/biopsy samples, Rhizopus arrhizus, R. homothallicus (n = 2); R. homothallicus, Lictheimia corymbifera (n = 1); R. arrhizus, Mucor spp (n = 1); and L. corymbifera, Apophysomyces variabilis (n = 1). All patients underwent surgical and medical (liposomal amphotericin B) treatment. All, except one growing A. variabilis and L. corymbifera survived. Mixed infection by more than one Mucorales in CAM is unique and warrants epidemiological investigation.