Investigations of an increased incidence of non-Aspergillus invasive mould infections in an onco-haematology unit.

AIMS OF THE STUDY: Invasive mould infections are life-threatening complications in patients with haematologic cancer and chemotherapy-induced neutropenia. While invasive aspergillosis represents the main cause of invasive mould infections, non-Aspergillus mould infections, such as mucormycosis, are increasingly reported. Consequently, their local epidemiology should be closely monitored. The aim of this study was to investigate the causes of an increased incidence of non-Aspergillus mould infections in the onco-haematology unit of a Swiss tertiary care hospital. METHODS: All cases of proven and probable invasive mould infections were retrospectively identified via a local registry for the period 2007-2021 and their incidence was calculated per 10,000 patient-days per year. The relative proportion of invasive aspergillosis and non-Aspergillus mould infections was assessed. Factors that may affect invasive mould infections' incidence, such as antifungal drug consumption, environmental contamination and changes in diagnostic approaches, were investigated. RESULTS: A significant increase of the incidence of non-Aspergillus mould infections (mainly mucormycosis) was observed from 2017 onwards (Mann and Kendall test p = 0.0053), peaking in 2020 (8.62 episodes per 10,000 patient-days). The incidence of invasive aspergillosis remained stable across the period of observation. The proportion of non-Aspergillus mould infections increased significantly from 2017 (33% vs 16.8% for the periods 2017-2021 and 2007-2016, respectively, p = 0.02). Building projects on the hospital site were identified as possible contributors of this increase in non-Aspergillus mould infections. However, novel diagnostic procedures may have improved their detection. CONCLUSIONS: We report a significant increase in non-Aspergillus mould infections, and mainly in mucormycosis infections, since 2017. There seems to be a multifactorial origin to this increase. Epidemiological trends of invasive mould infections should be carefully monitored in onco-haematology units in order to implement potential corrective measures.

Airway necrosis and granulation tissue formation caused by Rhizopus oryzae leading to severe upper airway obstruction: a case report.

Pulmonary Mucormycosis is a fatal infectious disease with high mortality rate. The occurrence of Mucormycosis is commonly related to the fungal virulence and the host's immunological defenses against pathogens. Mucormycosis infection and granulation tissue formation occurred in the upper airway was rarely reported. This patient was a 60-year-old male with diabetes mellitus, who was admitted to hospital due to progressive cough, sputum and dyspnea. High-resolution computed tomography (HRCT) and bronchoscopy revealed extensive tracheal mucosal necrosis, granulation tissue proliferation, and severe airway stenosis. The mucosal necrotic tissue was induced by the infection of Rhizopus Oryzae, confirmed by metagenomic next-generation sequencing (mNGS) in tissue biopsy. This patient was treated with the placement of a covered stent and local instillation of amphotericin B via bronchoscope. The tracheal mucosal necrosis was markedly alleviated, the symptoms of cough, shortness of breath, as well as exercise tolerance were significantly improved. The placement of airway stent and transbronchial microtube drip of amphotericin B could conduce to rapidly relieve the severe airway obstruction due to Mucormycosis infection.

Molecular mechanisms that govern infection and antifungal resistance in Mucorales.

SUMMARYThe World Health Organization has established a fungal priority pathogens list that includes species critical or highly important to human health. Among them is the order Mucorales, a fungal group comprising at least 39 species responsible for the life-threatening infection known as mucormycosis. Despite the continuous rise in cases and the poor prognosis due to innate resistance to most antifungal drugs used in the clinic, Mucorales has received limited attention, partly because of the difficulties in performing genetic manipulations. The COVID-19 pandemic has further escalated cases, with some patients experiencing the COVID-19-associated mucormycosis, highlighting the urgent need to increase knowledge about these fungi. This review addresses significant challenges in treating the disease, including delayed and poor diagnosis, the lack of accurate global incidence estimation, and the limited treatment options. Furthermore, it focuses on the most recent discoveries regarding the mechanisms and genes involved in the development of the disease, antifungal resistance, and the host defense response. Substantial advancements have been made in identifying key fungal genes responsible for invasion and tissue damage, host receptors exploited by the fungus to invade tissues, and mechanisms of antifungal resistance. This knowledge is expected to pave the way for the development of new antifungals to combat mucormycosis. In addition, we anticipate significant progress in characterizing Mucorales biology, particularly the mechanisms involved in pathogenesis and antifungal resistance, with the possibilities offered by CRISPR-Cas9 technology for genetic manipulation of the previously intractable Mucorales species.

Use of the Mucorales qPCR on blood to screen high-risk hematology patients is associated with better survival.

Our objective was to determine whether the twice-weekly screening of high-risk hematology patients by Mucorales qPCR on serum affects the prognosis of mucormycosis. Results from all serum Mucorales qPCR tests performed on patients from the hematology unit from January 2017 to December 2022 were analyzed. Patients with positive results were classified as having proven, probable or 'PCR-only' mucormycosis. One-month mortality for the local cohort was compared with that of a national cohort of cases of mucormycosis collected by the French surveillance network for invasive fungal disease ('Réseau de surveillances des infections fongiques invasives en France' (RESSIF)) from 2012 to 2018. From 2017 to 2022, 7825 serum Mucorales qPCR tests were performed for patients from the hematology unit; 107 patients with at least one positive Mucorales qPCR (164 positive samples) were identified. Sixty patients (70 positive samples, median Cq = 40) had no radiological criteria for mucormycosis and were considered not to have invasive fungal disease (70/7825, 0.9% false positives). It was not possible to classify disease status for six patients (12 positive samples, median Cq = 38). Forty-one patients (82 positive samples, median Cq = 35) had a final diagnosis of mucormycosis. In comparison with the RESSIF cohort, the local cohort was independently associated with a 48% lower one-month all-cause mortality rate (age-, sex-, and primary disease-adjusted hazard ratio = 0.52; 95% confidence interval: 0.29-0.94; P 0.03). Proactive screening for invasive mold diseases in high-risk hematology patients, including twice-weekly Mucorales qPCR on serum, was associated with mucormycosis higher survival.

Dissecting the genome sequence of a clinical isolated Cunninghamella bertholletiae Z2 strain with rich cytochrome P450 enzymes (Article).

Mucormycosis is receiving much more attention because of its high morbidity and extremely high mortality rate in immunosuppressed populations. In this study, we isolated a Cunnignhamella bertholletiae Z2 strain from a skin lesion of a 14 year, 9 months old girl with acute lymphoblastic leukemia who die of infection from the Z2 strain. Genome sequencing was performed after isolation and amplification of the Z2 strain to reveal potential virulence factors and pathogenic mechanisms. The results showed that the genome size of the Z2 strain is 30.9 Mb with 9213 genes. Mucoral specific virulence factor genes found are ARF, CalN, and CoTH, while no gliotoxin biosynthesis gene cluster was found, which is a known virulence factor in Aspergillus fumigatus adapted to the environment. The Z2 strain was found to have 69 cytochrome P450 enzymes, which are potential drug resistant targets. Sensitivity testing of Z2 showed it was only inhibited by amphotericin B and posaconazole. Detailed genomic information of the C. bertholletiae Z2 strain may provide useful data for treatment.

Role of Amphotericin B in the Treatment of Mucormycosis.

BACKGROUND: Regardless of the most recent inclusion of mold-active agents (isavuconazole and posaconazole) to antifungal agents against mucormycosis, in conjunction with amphotericin B (AMB) items, numerous uncertainties still exist regarding the treatment of this rare infection. The order Mucorales contains a variety of fungi that cause the serious but uncommon fungal illness known as mucormycosis. The moulds are prevalent in nature and typically do not pose significant risks to people. Immunocompromised people are affected by it. OBJECTIVE: This article's primary goal is to highlight the integral role that AMB plays in this condition. METHODS: Like sinusitis (including pansinusitis, rhino-orbital, or rhino-cerebral sinusitis) is one of the many signs and symptoms of mucormycosis. The National Center for Biotechnology Information (NCBI) produces a variety of online information resources for review articles on the topic-based mucormycosis, AMB, diagnosis of mucormycosis and the PubMed® database of citations and abstracts published in life science journals. These resources can be accessed through the NCBI home page at https://www.ncbi.nlm.nih.gov. RESULTS: The article provides a summary of the pharmacological attributes of the various AMB compositions accessible for systemic use. CONCLUSION: The article demonstrates the traits of the drug associated with its chemical, pharmacokinetics, stability, and other features, and illustrates their most useful characteristics for clinical application.

The mutual value of histopathology and ITS sequencing in the diagnosis of mucormycosis.

AIMS: Mucormycosis is a fast-progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis. Although histopathology often recognises invasive mould infections at first, histomorphology alone is insufficient in providing an accurate diagnosis. Unbiased molecular methods to detect and identify fungi are promising, yet their role in complementing routine histopathological workflows has not been studied sufficiently. METHODS AND RESULTS: We performed a retrospective single-centre study examining the clinical value of complementing histopathology with internal transcribed spacer (ITS) sequencing of fungal DNA in the routine diagnosis of mucormycosis. At our academic centre, we identified 14 consecutive mucormycosis cases diagnosed by histopathology and subsequent ITS sequencing. Using histomorphological examination, fungal hyphae could be detected in all cases; however, morphological features were unreliable regarding specifying the taxa. Subsequent ITS sequencing identified a remarkable phylogenetic diversity among Mucorales: the most common species was Rhizopus microsporus (six of 14; 42.9%), followed by Lichtheimia corymbifera (three of 14, 21.4%) and single detections of Rhizopus oryzae, Actinomucor elegans, Mucor circinelloides, Rhizomucor pusillus and Rhizomucor miehei (one of 14; 7.1%, respectively). In one case, we additionally detected Pneumocystis jirovecii in the same lung tissue specimen, suggesting a clinically relevant co-infection. Fungal culture was performed in 10 cases but yielded positive results in only two of 10 (20%), revealing its limited value in the diagnosis of mucormycosis. CONCLUSIONS: Our study demonstrates that a combination of histopathology and ITS sequencing is a practically feasible approach that outperforms fungal culture in detecting Mucorales in tissue-associated infections. Therefore, pathologists might adapt diagnostic workflows accordingly when mucormycosis is suspected.

Immune and metabolic perturbations in COVID-19-associated pulmonary mucormycosis: A transcriptome analysis of innate immune cells.

BACKGROUND AND OBJECTIVES: The mechanisms underlying COVID-19-associated pulmonary mucormycosis (CAPM) remain unclear. We use a transcriptomic analysis of the innate immune cells to investigate the host immune and metabolic response pathways in patients with CAPM. PATIENTS AND METHODS: We enrolled subjects with CAPM (n = 5), pulmonary mucormycosis (PM) without COVID-19 (n = 5), COVID-19 (without mucormycosis, n = 5), healthy controls (n = 5) without comorbid illness and negative for SARS-CoV-2. Peripheral blood samples from cases were collected before initiating antifungal therapy, and neutrophils and monocytes were isolated. RNA sequencing was performed using Illumina HiSeqX from monocytes and neutrophils. Raw reads were aligned with HISAT-2 pipeline and DESeq2 was used for differential gene expression. Gene ontology (GO) and metabolic pathway analysis were performed using Shiny GO application and R packages (ggplot2, Pathview). RESULTS: The derangement of core immune and metabolic responses in CAPM patients was noted. Pattern recognition receptors, dectin-2, MCL, FcRγ receptors and CLEC-2, were upregulated, but signalling pathways such as JAK-STAT, IL-17 and CARD-9 were downregulated; mTOR and MAP-kinase signalling were elevated in monocytes from CAPM patients. The complement receptors, NETosis, and pro-inflammatory responses, such as S100A8/A9, lipocalin and MMP9, were elevated. The major metabolic pathways of glucose metabolism-glycolysis/gluconeogenesis, pentose phosphate pathway, HIF signalling and iron metabolism-ferroptosis were also upregulated in CAPM. CONCLUSIONS: We identified significant alterations in the metabolic pathways possibly leading to cellular iron overload and a hyperglycaemic state. Immune responses revealed altered recognition, signalling, effector functions and a pro-inflammatory state in monocytes and neutrophils from CAPM patients.

Mucorales fungi suppress nitric oxide production by macrophages.

IMPORTANCE: In October 2022, Mucorales fungi were listed in the "High Priority Group" on the first-ever list of fungal priority pathogens by the World Health Organization. As the causative agent of mucormycosis, Mucorales have become of great clinical and public health importance with growing mucormycosis numbers, notably with the exponential rise of COVID-19-associated mucormycosis cases. Despite the dire need, there are limited therapeutic options to treat mucormycosis. Our research fills in critical gaps of knowledge about how Mucorales fungi evade the host immune system. Specifically, we offer evidence that Mucorales block nitric oxide production, which is a key mediator and signaling molecule of the mammalian innate immune response to microbial pathogens. Our work offers new insight into immune evasion mechanisms by Mucorales fungi.

Antifungal Activity of Chitosan Polymeric Nanoparticles and Correlation with Their pH Against Mucor circinelloides Causing Mucormycosis, Along with Penicillium notatum and Aspergillus Species.

Mucormycosis is uncommon, yet it is more prevalent among individuals with underlying health conditions and those who are immunocompromised. Chitosan is studied because of its appealing properties and diverse applications. The purpose of this work is to synthesize chitosan nanoparticles (CSNPs) by ionic gelation method at various pH levels and test them against Mucor and other filamentous fungus. Field Emission Scanning Electron Microscope, Zeta sizer, Zeta potential, and Fourier Transformed Infrared Spectroscopy were used to characterize CSNPs. Hydrodynamic size increased considerably with increasing pH. Our CSNPs were tested against fungal isolates of Aspergillus Flavus RCMB 02783, Aspergillus Fumigatus RCMB 02564, and Aspergillus Niger RCMB 02588, Penicillium Notatum (NCPF 2881) and   (M. circinelloides CNRMA 03.894) causing mucromycosis. Antifungal activity was investigated using Minimum inhibitory concentration (MIC), Minimum Fungicidal concentration (MFC), Disc diffusion assay, and Antifungal inhibitory percentages methods. The best antifungal efficacy results were obtained through CSNPs prepared at pH = 4.4 at very low concentration for MIC (1.03 or 2.75 µg/mL) with 100% M. circinelloides inhibition followed by pH = 4.6 with MIC (73 or 208 µg/mL) and 93%  M. cirecinelloides inhibition %. Future usage of these materials in masks or wound dressing to avoid fungal infections, including mucormycosis following COVID-19, penicillium, and aspergillosis toxicity and infections.

Mucorales-disseminated infection in burn wound.

A previously fit and well man in his 50s was rescued from a burning apartment with Glasgow Coma Scale 3 and admitted to the burn intensive care unit with 18% mixed dermal and full thickness burns and inhalation injury. He received standardised acute burn treatment according to the Emergency Management of Severe Burn guidelines and was found to have acute kidney injury requiring dialysis and cerebral watershed infarcts. The burns were deep especially on the left leg that was deemed unsalvageable and on day 8, he underwent a mid-femoral amputation.A wound swab on day 8 grew mould and with progression of skin necrosis, Mucorales infection was clinically suspected. Microbiological assessment of the swab confirmed Mucorales infection-an invasive fungus with the ability to invade blood vessels leading to vessel thrombosis and tissue necrosis and associated with high mortality. Recommended radical debridement with free cutaneous margins was not possible due to the widespread disease, and the patient was treated conservatively with antifungal therapy and survived.

Whole genome analysis of Rhizopus species causing rhino-cerebral mucormycosis during the COVID-19 pandemic.

Introduction: Mucormycosis is an acute invasive fungal disease (IFD) seen mainly in immunocompromised hosts and in patients with uncontrolled diabetes. The incidence of mucormycosis increased exponentially in India during the SARS-CoV-2 (henceforth COVID-19) pandemic. Since there was a lack of data on molecular epidemiology of Mucorales causing IFD during and after the COVID-19 pandemic, whole genome analysis of the Rhizopus spp. isolated during this period was studied along with the detection of mutations that are associated with antifungal drug resistance. Materials and methods: A total of 50 isolates of Rhizopus spp. were included in this prospective study, which included 28 from patients with active COVID-19 disease, 9 from patients during the recovery phase, and 13 isolates from COVID-19-negative patients. Whole genome sequencing (WGS) was performed for the isolates, and the de novo assembly was done with the Spades assembler. Species identification was done by extracting the ITS gene sequence from each isolate followed by searching Nucleotide BLAST. The phylogenetic trees were made with extracted ITS gene sequences and 12 eukaryotic core marker gene sequences, respectively, to assess the genetic distance between our isolates. Mutations associated with intrinsic drug resistance to fluconazole and voriconazole were analyzed. Results: All 50 patients presented to the hospital with acute fungal rhinosinusitis. These patients had a mean HbA1c of 11.2%, and a serum ferritin of 546.8 ng/mL. Twenty-five patients had received steroids. By WGS analysis, 62% of the Rhizopus species were identified as R. delemar. Bayesian analysis of population structure (BAPS) clustering categorized these isolates into five different groups, of which 28 belong to group 3, 9 to group 5, and 8 to group 1. Mutational analysis revealed that in the CYP51A gene, 50% of our isolates had frameshift mutations along with 7 synonymous mutations and 46% had only synonymous mutations, whereas in the CYP51B gene, 68% had only synonymous mutations and 26% did not have any mutations. Conclusion: WGS analysis of Mucorales identified during and after the COVID-19 pandemic gives insight into the molecular epidemiology of these isolates in our community and establishes newer mechanisms for intrinsic azole resistance.

Advancements of fish-derived peptides for mucormycosis: a novel strategy to treat diabetic compilation.

Mucormycosis, an extremely fatal fungal infection, is a major hurdle in the treatment of diabetes consequences. The increasing prevalence and restricted treatment choices urge the investigation of novel therapeutic techniques. Because of their effective antimicrobial characteristics and varied modes of action, fish-derived peptides have lately emerged as viable options in the fight against mucormycosis. This review examines the potential further application of fish-derived peptides in diagnosing and managing mucormycosis in relation to diabetic complications. First, we examine the pathophysiology of mucormycosis and the difficulties in treating it in diabetics. We emphasize the critical need for alternative therapeutic methods for tackling the limitations of currently available antifungal medicines. The possibility of fish-derived peptides as an innovative approach to combat mucormycosis is then investigated. These peptides, derived from several fish species, provide wide antimicrobial properties against a variety of diseases. They also have distinct modes of action, such as rupture of cell membranes, suppression of development, and modification of the host immunological response. Furthermore, we investigate the problems and prospects connected with the clinical application of fish-derived peptides. Ultimately, future advances in fish-derived peptides, offer interesting avenues for the management of mucormycosis in the context of diabetic comorbidities. More research and clinical trials are needed to properly investigate these peptide's therapeutic potential and pave the way for their adoption into future antifungal therapies.

Case Report and Literature Review of Prosthetic Cardiovascular Mucormycosis.

We report a rare case of aorto-bi-iliac prosthetic allograft mucormycosis in a 57-year-old immunocompetent patient in France. Outcome was favorable after surgery and dual antifungal therapy with liposomal amphotericin B and isavuconazole. In a literature review, we identified 12 other cases of prosthetic vascular or heart valve mucormycosis; mortality rate was 38%.

Comparative evaluation of histopathological analysis, KOH wet mount and fungal culture to diagnose fungal infections in post-COVID patients.

Context and Aim: There is increasing prevalence of post-COVID fungal infection of rhinoorbitocerebral region especially mucormycosis and aspergillosis in India.[1] Early diagnosis of these fungal infections are of utmost importance, since it may improve outcome and survival.[2],[3],[4],[5],[6],[7],[8] The objective of this study was to evaluate and compare routine laboratory diagnostic methods, that is, histopathological examination, KOH wet mount and fungal culture in the diagnosis of post-COVID fungal infections. Materials and Methods: A total of 106 specimens of clinically suspected patients of post-COVID fungal infection of rhinoorbitocerebral region received in histopathology department were included in this study. The data of KOH wet mount and culture were acquired from the microbiology department after histopathological examination. Result: Approximately 88.68% of patients were diagnosed having fungal infections by one of the laboratory methods. The sensitivity of histopathological examination was highest (79.78%), followed by KOH wet mount (58.51%) and fungal culture (35.10%). Rhizopus species of zygomycetes group were the most common isolate (24.24%) on SDA culture. Overall 76% concordance was found between histopathological examination and fungal culture report for morphological identification of fungi. Conclusion: For the diagnosis of post-COVID fungal infection of Rhino-orbito-cerebral region, histopathological examination is was found to be more sensitive and rapid method to detect fungal hyphae. It leads to early treatment, prevents morbidity and mortality.

Local injection of amphotericin B: novel use in the treatment of fungal maxillary sinusitis.

This report highlights the role of local amphotericin B (AMB) injection in cases of maxillary mucormycosis. The COVID-19 pandemic has resulted in a dramatic rise in the number of rhino-orbital mucormycosis cases. Although extensive surgical debridement remains the gold standard treatment, tissue salvage is desirable. The cases of two patients treated with local AMB are reported here, indicating that early intervention for maxillary fungal sinusitis in the form of local AMB may avoid the need for more invasive treatment.

Post-COVID-19 rhino-orbito-cerebral mucormycosis-A clinico-mycological study from North India.

The second wave of coronavirus disease 2019 (COVID-19), during the early 2021, lead to a devastating outbreak of mucormycosis in India. This study aimed to determine the aetiology, clinical features, comorbidities, and risk factors of rhino-orbito-cerebral mucormycosis (ROCM) and antifungal susceptibility pattern for the isolates. The study included all suspected cases of ROCM in post-COVID-19 patients attending the hospital from May to December 2021. A total of 70 patients were diagnosed with mucormycosis during the study period. The commonest presentations were rhino-orbital and rhino-orbito-cerebral in 35.7% of cases each. Diabetes mellitus was the commonest associated risk factor in 95.7% of all patients, while 78.5% of the patients were treated with corticosteroids in the recent past, and 25.7% presented with active COVID-19 pneumonia. The commonest isolate was Rhizopus arrhizus n = 14, followed by Aspergillus flavus n = 16, A. fumigatus n = 4, A. niger n = 3, Fusarium oxysporumn = 1, and Apophysomyces variabilisn = 1. Fungal species identification was done by phenotypic methods for all the isolates and DNA sequence analysis of 18 isolates, and antifungal susceptibility testing of 30 isolates was performed by commercially prepared HiMIC plate (HiMedia, Mumbai, India) using broth microdilution for amphotericin B, isavuconazole, itraconazole, voriconazole, and posaconazole. The MIC50 and MIC90 of amphotericin B for R. arrhizus strains were 0.25 and 4 µg/ml, respectively; and the MIC50 and MIC90 results for itraconazole, posaconazole, and isavuconazole were 8 and 8, 2 and 2, and 2 and 8 µg/ml, respectively. In vitro data showed that amphotericin B was the most effective antifungal against most species. The commercially available ready-to-use minimum inhibitory concentration plates are user-friendly for performing antifungal susceptibility, which may be useful in choosing appropriate regimens and monitoring emerging resistance.

Lack of surgical resection is associated with increased early mortality in hematological patients complicated with rhino-orbital-cerebral mucormycosis.

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.