A case of COVID-19-associated mucormycosis due to Lichtheimia ramosa.

BACKGROUND: Mucormycosis is a life-threatening invasive fungal infection in immunocompromised and COVID-19 patients. CASE REPORT: Here, we report a fatal rhino-orbito-cerebral mucormycosis caused by Lichtheimia ramosa, in a 79-year-old diabetic female. She was initially admitted to the hospital for COVID-19 infection and received broad-spectrum antibiotics and corticosteroids. After 1 month, she was admitted again because of persistent headaches and decreased right eye movement when the computed tomography scan showed mucosal thickening and opacification of paranasal sinuses. Microbiological investigations, including culture and direct microscopy, and histopathological findings confirmed the diagnosis of proven mucormycosis. The isolated causal agent was identified as Lichtheimia ramosa by sequencing the entire ITS region of nuclear ribosomal DNA. Despite surgical debridement and administration of liposomal amphotericin B 5 mg/kg/day, the patient's level of consciousness suddenly deteriorated; she was intubated and mechanically ventilated in the ICU and died on the same day. CONCLUSION: To our knowledge, this is the first worldwide case of COVID-19-associated rhino-orbito-cerebral mucormycosis due to Lichtheimia ramosa.

A new terrein dimer and a new meroterpene from the mangrove endophytic fungus Lichtheimia sp.

A new terrein dimer named lichtheicol A (1) and a new meroterpene named lichtheiterpene A (2), were isolated from the mangrove endophytic fungus Lichtheimia sp. J2B1, together with 10 known compounds (3-12). The planar structures and absolute configurations of 1 and 2 were established by a combination of extensive spectroscopic data analyses and electronic circular dichroism (ECD) calculations. Compounds 4 and 5 exhibited marked inhibitory effects against butyrylcholinesterase (BuChE) with IC50 values of 0.71 and 0.53 µM, respectively.

Parietal composition of Lichtheimia corymbifera: Differences between spore and germ tube stages and host-pathogen interactions.

The molecular composition and structural organization of the cell wall of filamentous fungi underlie the ability of the host to identify them as pathogens. Although the organization of the fungal cell wall, composed of 90% polysaccharides, is similar from one fungus to another, small variations condition their ability to trigger pattern recognition receptors. Because the incidence of mucormycosis, an emerging life-threatening infection caused by the species of the order Mucorales is increasing worldwide, the precise composition of the cell wall of two strains of Lichtheimia corymbifera was investigated in the early growth stages of germination (spores and germ-tubes) using trimethylsilylation and confocal microscopy. This study also characterizes the response of THP-1 cells to Mucorales. The study identified the presence of uncommon monosaccharides (fucose, galactose, and glucuronic acid) whose respective proportions vary according to the germination stage, revealing early parietal reorganization. Immunofluorescence studies confirmed the exposure of ß-glucan on the surface of swollen spores and germ-tubes. Both spores and germ-tubes of L. corymbifera promoted an early and strong pro-inflammatory response, through TLR-2. Our results show the singularity of the cell wall of the order Mucorales, opening perspectives for the development of specific diagnostic biomarkers.

Lichtheimia corymbifera is a causative agent of mucormycosis, an emerging invasive fungal infection. Deciphering cell wall composition can lead to the identification of a polysaccharide epitope, which could be used as a biomarker, useful for the diagnosis of mucormycosis.

Characterization of mitogenomes from four Mucorales species and insights into pathogenicity.

BACKGROUND: Mucorales, as one major order of Zygomycetes fungi, can infect human beings and cause serious consequence. We have noticed the pathogenicity of Mucorales is closely related to energy metabolism, while mitochondria play the role of energy factories in almost all biological activities. METHODS: Virulence of M irregularis, M hiemalis, L corymbifera and R arrhizus were verified in Galleria mellonella larvae, as well as mitochondrial gene copies analysed with RT-qPCR. Mitogenomes of the four Mucorales species were sequenced based on illumina NovaSeq technology to study their characteristic features and functional regions. RESULTS: Variant virulence of M irregularis, M hiemalis, L corymbifera and R arrhizu were verified by clinical retrospective data and our G mellonella infection models, also copies of mitochondrial genes indicated the significant associations with pathogenicity. A total of 274.18 clean reads were generated to be assembled; the complete mitogenomes of the four Mucorales species were obtained with totally different length. After the genomes annotated and compared, M irregularis was found more similar with M hiemalis than those of L corymbifera and R arrhizus, especially the small (rrns) and large (rrnl) subunits of mitochondrial ribosomal RNA (rRNA) genes. The GC content, ncRNAs and the distribution of the SNPs and InDels were also compared, and the GC content rate of fungi seems to be related to the fungal thermal adaptability. In addition, linear mitogenomes of these four Mucorales showed diverse arrangements of orf genes and directionality of some conserved gene elements. CONCLUSION: This study uncovered the pathogenicity variances among the four Mucorales species and the relationship between their mitogenomic features and clinical pathogenicity. Further studies like spatial structure of mitochondrial genomes and the comprehensive analysis of transcription regulation are needed.

Corticosteroids alter alveolar macrophage control of Lichtheimia corymbifera spores in an ex vivo mouse model.

Alveolar macrophages (AM) are the first-line lung defense against Mucorales in pulmonary mucormycosis. Since corticosteroid use is a known risk factor for mucormycosis, the aim of this study was to describe the role of corticosteroids on AM capacities to control Lichtheimia corymbifera spore growth using a new ex vivo model. An in vivo mouse model was developed to determine the acetate cortisone dose able to trigger pulmonary invasive infection. Then, in the ex vivo model, male BALB/c mice were pretreated with the corticosteroid regimen triggering invasive infection, before AM collection through bronchoalveolar lavage. AMs from corticosteroid-treated mice and untreated control AMs were then exposed to L. corymbifera spores in vitro (ratio 1:5). AM control of fungal growth, adherence/phagocytosis, and oxidative burst were assessed using optical densities by spectrophotometer, flow cytometry, and 2', 7'-dichlorofluoresceine diacetate fluorescence, respectively. Cortisone acetate at 500 mg/kg, at D-3 and at D0, led to pulmonary invasive infection at D3. Co-incubated spores and AMs from corticosteroid-treated mice had significantly higher absorbance (fungal growth) than co-incubated spores and control AMs, at 24 h (P = .025), 36 h (P = .004), and 48 h (P = .001). Colocalization of spores with AMs from corticosteroid-treated mice was significantly lower than for control AMs (7.6 ± 1.9% vs 22.3 ± 5.8%; P = .003), reflecting spore adherence and phagocytosis inhibition. Finally, oxidative burst was significantly increased when control AMs were incubated with spores (P = 0.029), while corticosteroids hampered oxidative burst from treated AMs (P = 0.321). Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease in our ex vivo model. LAY SUMMARY: The aim of this study was to describe the impact of corticosteroids on alveolar macrophage (AM) capacities to control Mucorales growth in a new murine ex vivo model. Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease.

Outbreaks of Mucorales and the Species Involved.

The order Mucorales is an ancient group of fungi classified in the subphylum Mucoromycotina. Mucorales are mainly fast-growing saprotrophs that belong to the first colonizers of diverse organic materials and represent a permanent part of the human environment. Several species are able to cause human infections (mucormycoses) predominantly in patients with impaired immune system, diabetes, or deep trauma. In this review, we compiled 32 reports on community- and hospital-acquired outbreaks caused by Mucorales. The most common source of mucoralean outbreaks was contaminated medical devices that are responsible for 40.7% of the outbreaks followed by contaminated air (31.3%), traumatic inoculation of soil or foreign bodies (9.4%), and the contact (6.2%) or the ingestion (6.2%) of contaminated plant material. The most prevalent species were Rhizopus arrhizus and R. microsporus causing 57% of the outbreaks. The genus Rhizomucor was dominating in outbreaks related to contaminated air while outbreaks of Lichtheimia species and Mucor circinelloides were transmitted by direct contact. Outbreaks with the involvement of several species are reported. Subtyping of strains revealed clonality in two outbreaks and no close relation in two other outbreaks. Based on the existing data, outbreaks of Mucorales can be caused by heterogeneous sources consisting of different strains or different species. Person-to-person transmission cannot be excluded because Mucorales can sporulate on wounds. For a better understanding and prevention of outbreaks, we need to increase our knowledge on the physiology, ecology, and population structure of outbreak causing species and more subtyping data.

Widespread Lichtheimia Infection in a Patient with Extensive Burns: Opportunities for Novel Antifungal Agents.

The Mucorales fungi-formerly classified as the zygomycetes-are environmentally ubiquitous fungi, but generally rare causes of clinical infections. In the immunocompromised host, however, they can cause invasive, rapidly spreading infections that confer a high risk of morbidity and mortality, often despite surgical and antifungal therapy. Patients with extensive burn injuries are particularly susceptible to skin and soft-tissue infections with these organisms. Here, we present a case of Lichtheimia infection in a patient with extensive full-thickness burns that required significant and repeated surgical debridement successfully treated with isavuconazole and adjunctive topical amphotericin B washes. We also review the available literature on contemporary antifungal treatment for Lichtheimia species and related Mucorales fungi.

Rhinocerebral Zygomycosis Due to a Lichtheimia ramosa Infection in a Calf: Neural Spread Through the Olfactory Nerves.

Here, we report a case of rhinocerebral zygomycosis due to a Lichtheimia ramosa infection in a calf. A histopathological examination revealed that a fungus had invaded the brain through the olfactory nerves. Lichtheimia ramosa was detected by polymerase chain reaction analysis of DNA extracted from formalin-fixed paraffin-embedded samples of the affected tissue. This is the first case of rhinocerebral zygomycosis to involve cattle. Also, this is the first such case to involve fungal invasion into the central nervous system through the cranial nerve itself, rather than through perineural tissue.

PCR-Based Approach Targeting Mucorales-Specific Gene Family for Diagnosis of Mucormycosis.

Mucormycosis is an aggressive, life-threatening infection caused by fungi in the order Mucorales. The current diagnosis of mucormycosis relies on mycological cultures, radiology and histopathology. These methods lack sensitivity and are most definitive later in the course of infection, resulting in the prevention of timely intervention. PCR-based approaches have shown promising potential in rapidly diagnosing mucormycosis. The spore coating protein homolog encoding CotH genes are uniquely and universally present among Mucorales. Thus, CotH genes are potential targets for the rapid diagnosis of mucormycosis. We infected mice with different Mucorales known to cause human mucormycosis and investigated whether CotH could be PCR amplified from biological fluids. Uninfected mice and those with aspergillosis were used to determine the specificity of the assay. CotH was detected as early as 24 h postinfection in plasma, urine, and bronchoalveolar lavage (BAL) samples from mice infected intratracheally with Rhizopus delemar, Rhizopus oryzae, Mucor circinelloides, Lichtheimia corymbifera, or Cunninghamella bertholletiae but not from samples taken from uninfected mice or mice infected with Aspergillus fumigatus Detection of CotH from urine samples was more reliable than from plasma or BAL fluid. Using the receiver operating characteristic method, the sensitivity and the specificity of the assay were found to be 90 and 100%, respectively. Finally, CotH was PCR amplified from urine samples of patients with proven mucormycosis. Thus, PCR amplification of CotH is a promising target for the development of a reliable, sensitive, and simple method of early diagnosis of mucormycosis.

Comparative analysis of nonvolatile and volatile metabolites in Lichtheimia ramosa cultivated in different growth media.

Lichtheimia ramosa is one of the predominant filamentous fungi in Korean traditional nuruk. The nonvolatile and volatile metabolites of L. ramosa cultivated in three growth media: complete medium (CM), potato dextrose broth (PDB), and sabouraud dextrose broth (SDB), were investigated and compared. Among nonvolatile metabolites, serine, lysine, and ornithine increased in CM and PDB cultivated with L. ramosa during the exponential phase. In addition, glucose level increased in CM whereas decreased in PDB and SDB. The major volatile metabolites in the extract samples were acetic acid, ethanol, 3-methyl-2-buten-1-ol, 2-phenylethanol, ethylacetate, 2-furaldehyde, 5-(hydroxymethyl)-2-furaldehyde, 2,3-dihydro-3,5,-dihydroxy-6-methyl-4H-pyran-4-one, and α-humulene. In particular, the levels of volatile metabolites related to makgeolli (e.g., acetic acid, ethanol, and ethyl acetate) were highest in extracts cultivated in CM. On the other hand, the level of 2-phenylethanol was relatively higher in PDB and SDB, possibly due to there being more phenylalanine present in the biomass sample in media.

Coinfection with Lichtheimia corymbifera and Aspergillus flavus in an Immune-Competent Patient Mimicking as Pulmonary-Renal Syndrome.

Lichtheimia corymbifera and Aspergillus flavus pulmonary coinfection has been rarely reported in immune-competent patients. We report case of a young male who presented with clinical features of pulmonary-renal syndrome and was later diagnosed to have bilateral polymicrobial fungal lung infection.

Early Diagnosis of Cutaneous Mucormycosis Due to Lichtheimia corymbifera After a Traffic Accident.

A case report of cutaneous mucormycosis and obstacles to early diagnosis is presented. A 38-year-old male was involved in a car accident that led to amputation of both lower limbs. Subsequently, he developed fungal wound infection of the left lower limb stump. The infection was detected very early, although the diagnosis was difficult because only a small area was affected and histopathological examination was initially negative. The infection was proven by microscopy, culture and histopathology. The isolate was identified by sequencing of the rDNA ITS region gene (internal transcribed spacer region of ribosomal DNA) as Lichtheimia corymbifera. Liposomal amphotericin B and surgery were successful in management of the disease.

Mucormycoses caused by Lichtheimia species.

Mucormycoses are life-threatening infections with fungi from the order Mucorales (Mucoromycotina). Although mucormycoses are uncommon compared to other fungal infections, e.g. aspergillosis and candidiasis, the number of cases is increasing especially in immunocompromised patients. Lichtheimia (formerly Absidia) species represent the second to third most common cause of mucormycoses in Europe. This mini review presents current knowledge about taxonomy and clinical relevance of Lichtheimia species. In addition, clinical presentation and risk factors will be discussed. Proper animal infection models are essential for the understanding of the pathogenesis and the identification of virulence factors of fungal pathogens. To date, several animal models have been used to study Lichtheimia infection. A brief overview of the different models and the main conclusions from the infection experiments is summarised in this review.

The pathogenic potential of the Lichtheimia genus revisited: Lichtheimia brasiliensis is a novel, non-pathogenic species.

Lichtheimia brasiliensis was recently described as a novel species within the genus Lichtheimia, which comprises a total of six species. L. brasiliensis was first reported from soil in Brazil. The aim of the study was to determine the relative virulence potential of L. brasiliensis using an avian infection model based on chicken embryos.

Mucormycosis in haematological patients: case report and results of prospective study in Saint Petersburg, Russia.

We prospectively observed 36 haematological patients with mucormycosis from nine hospitals of St. Petersburg during 2004-2013. The most frequent underlying diseases were acute leukaemia (64%), and main risk factors were prolonged neutropenia (92%) and lymphocytopenia (86%). In 50% of the patients, mucormycosis was diagnosed 1-65 days after invasive aspergillosis. Main clinical form of mucormycosis was pulmonary (64%), while two or more organ involvement was noted in 50% of the cases. The most frequent aetiological agents of mucormycosis were Rhizopus spp. (48%). Twelve-week survival rate was 50%. Combination therapy (echinocandins + amphotericin B forms) and recovery from the underlying disease significantly improved the survival rate.

A Systematic Review of the Therapeutic Outcome of Mucormycosis.

Background: Mucormycosis is a potentially lethal mycosis. We reviewed peer-reviewed publications on mucormycosis to assess therapeutic outcomes. Methods: A systematic literature search using the Ovid MEDLINE and EMBASE databases identified manuscripts describing human mucormycosis diagnosed according to European Organization for Research and Treatment of Cancer and the Mycoses Study Group criteria with therapeutic outcomes published from 2000 to 2022. Results: In 126 articles, 10 335 patients were described, most from Asia (n = 6632, 66%). Diabetes was the most frequent underlying disease (n = 6188, 60%); 222 (2.1%) patients had no underlying diseases. The dominant clinical form was rhino-orbitocerebral (n = 7159, 69.3%), followed by pulmonary (n = 1062, 10.3%). Of 5364 patients with outcome data, amphotericin B monotherapy (n = 3749, mortality 31.5%) was most frequent, followed by amphotericin B + azole (n = 843, mortality 6.6%; P < .0001), amphotericin B followed by azole (n = 357, mortality 13.7%; P < .0001), posaconazole only (n = 250, mortality 17.2%; P < .0001), and isavuconazole only (n = 65, mortality 24.6%; P = .24). Duration and dose of antifungals varied widely. Documented outcomes from surgical resections in 149 patients found that 47 of 125 died (37.6%), compared with 16 of 24 (66.7%) patients who did not undergo surgery (P = .008). Conclusions: Mucormycosis is more frequently reported in Asia than in Europe and is often linked to diabetes. Antifungal therapy, usually with surgery, is frequently effective for mucormycosis.

Facial and systemic mucormycosis caused by Lichtheimia corymbifera in a goat: case report and literature review of fungal infections in goats.

An 8-y-old Pygora doe was presented to the University of California-Davis, Veterinary Medical Teaching Hospital because of non-healing facial swelling of 2-wk duration. The lesion grew despite medical treatment, causing discomfort masticating, little-to-no airflow from the right nasal passage, and led to euthanasia. On gross examination, a large facial mass with a draining tract through the skin and hard palate was identified. On section, the mass was brown-pink, homogeneous, and friable. Abscess-like masses were identified in the lungs and kidney. Histopathology of the face, including oral and nasal cavities, salivary glands, and lymph nodes, as well as the lung and kidney lesions, revealed large areas of necrosis with numerous wide ribbon-like, mostly aseptate, fungal hyphae consistent with zygomycetes. PCR for fungal organisms performed on formalin-fixed, paraffin-embedded tissue from the face identified Lichtheimia corymbifera (formerly Absidia corymbifera) of the order Mucorales and an Aspergillus sp. The lesion was suspected to have started either as a fungal rhinitis or dental feed impaction, subsequently spreading to the face and systemically to the lungs and kidney. We describe here the lesions associated with facial mucormycosis in a goat and present a literature review of L. corymbifera infection in veterinary species and fungal infections in goats.

Development of a Combat-Relevant Murine Model of Wound Mucormycosis: A Platform for the Pre-Clinical Investigation of Novel Therapeutics for Wound-Invasive Fungal Diseases.

Wound-invasive fungal diseases (WIFDs), especially mucormycosis, have emerged as life-threatening infections during recent military combat operations. Many combat-relevant fungal pathogens are refractory to current antifungal therapy. Therefore, animal models of WIFDs are urgently needed to investigate new therapeutic solutions. Our study establishes combat-relevant murine models of wound mucormycosis using Rhizopus arrhizus and Lichtheimia corymbifera, two Mucorales species that cause wound mucormycosis worldwide. These models recapitulate the characteristics of combat-related wounds from explosions, including blast overpressure exposure, full-thickness skin injury, fascial damage, and muscle crush. The independent inoculation of both pathogens caused sustained infections and enlarged wounds. Histopathological analysis confirmed the presence of necrosis and fungal hyphae in the wound bed and adjacent muscle tissue. Semi-quantification of fungal burden by colony-forming units corroborated the infection. Treatment with liposomal amphotericin B, 30 mg/kg, effectively controlled R. arrhizus growth and significantly reduced residual fungal burden in infected wounds (p < 0.001). This study establishes the first combat-relevant murine model of wound mucormycosis, paving the way for developing and evaluating novel antifungal therapies against combat-associated WIFDs.

Mucormycosis as a rare cause of severe gastrointestinal bleeding after multivisceral transplantation.

Mucormycosis, an emerging fungal infection in solid organ transplant patients, is mostly located in rhino-orbito-cerebral, pulmonary, and cutaneous areas, or disseminated with poor prognosis. A 4-year-old girl with chronic intestinal pseudo-obstruction syndrome underwent a modified multivisceral transplantation, including half of the stomach, the duodeno-pancreas, the small bowel, and the right colon. On postoperative day 5, a digestive perforation was suspected. Surgical exploration found a small necrotic area on the native stomach, which was externally drained. The next day, massive gastric bleeding occurred. During the emergency laparotomy, 2 hemorrhagic ulcers were found and resected from the transplanted stomach. Pathology and fungal culture showed mucormycosis caused by Lichtheimia (formerly Absidia) ramosa in both the transplanted and native stomach. High-dose intravenous liposomal amphotericin B was immediately started. No other site of fungal infection was found. The child recovered, and 3 years after transplantation, is alive and well, off parenteral nutrition. The originality of this case is the very early presentation after transplantation, the unusual site, and the complete recovery after rapid medico-surgical management. The origin of the fungus and treatment are discussed.

Antifungal Activity of Isavuconazole and Comparator Agents against Contemporaneous Mucorales Isolates from USA, Europe, and Asia-Pacific.

Isavuconazole is the only US FDA-approved antifungal for treating invasive mucormycosis. We evaluated isavuconazole activity against a global collection of Mucorales isolates. Fifty-two isolates were collected during 2017-2020 from hospitals located in the USA, Europe, and the Asia-Pacific. Isolates were identified by MALDI-TOF MS and/or DNA sequencing and susceptibility tested by the broth microdilution method following CLSI guidelines. Isavuconazole (MIC50/90, 2/>8 mg/L) inhibited 59.6% and 71.2% of all Mucorales isolates at ≤2 mg/L and ≤4 mg/L, respectively. Among comparators, amphotericin B (MIC50/90, 0.5/1 mg/L) displayed the highest activity, followed by posaconazole (MIC50/90, 0.5/8 mg/L). Voriconazole (MIC50/90, >8/>8 mg/L) and the echinocandins (MIC50/90, >4/>4 mg/L) had limited activity against Mucorales isolates. Isavuconazole activity varied by species and this agent inhibited at ≤4 mg/L 85.2%, 72.7%, and 25% of Rhizopus spp. (n = 27; MIC50/90, 1/>8 mg/L), Lichtheimia spp. (n = 11; MIC50/90, 4/8 mg/L), and Mucor spp. (n = 8; MIC50, >8 mg/L) isolates, respectively. Posaconazole MIC50/90 values against Rhizopus, Lichtheimia, and Mucor species were 0.5/8 mg/L, 0.5/1 mg/L, and 2/- mg/L, respectively; amphotericin B MIC50/90 values were 1/1 mg/L, 0.5/1 mg/L, and 0.5/- mg/L, respectively. As susceptibility profiles varied among Mucorales genera, species identification and antifungal susceptibility testing are advised whenever possible to manage and monitor mucormycosis.