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.

Primary cutaneous mucormycosis due to Rhizopus arrhizus in an immunosuppressed patient with paroxysmal nocturnal haemoglobinuria.

OBJECTIVE: We present a case of primary cutaneous mucormycosis in a patient with bone marrow failure secondary to paroxysmal nocturnal haemoglobinuria (PNH). CLINICAL CASE: A 60-year-old male patient with a history of PNH, complicated to a severe aplastic anaemia, presented to the emergency department complaining of papules on the lower limbs that rapidly turned into necrotic plaques within 2 months. Histopathological examination showed granulomatous and suppurative dermatitis with tissue necrosis and the presence of non-septate hyphae. Molecular identification was achieved by amplification and sequencing of the 18S-ITS1-5.8S-ITS2-28S rRNA region using the polymerase chain reaction. The sequence showed 100% identity with Rhizopus arrhizus. The patient received treatment with liposomal amphotericin B and surgical debridement. Nonetheless, the patient suffered from severe low red blood cells and platelets and also underwent septic shock; he died 6 days after admission to the hospital. CONCLUSION: Mucormycosis in the setting of immunosuppression is challenging. Upon suspicion of a diagnosis, immediate treatment is required. Adjunctive therapies may be considered; however, the case fatality rate remains high.

Mucormycosis in Africa: Epidemiology, diagnosis and treatment outcomes.

Mucorales fungi cause mucormycosis, an invasive and rapidly progressive disease which increasingly affects mostly immunocompromised but also immunocompetent individuals. The objective of this study was to highlight the epidemiology, diagnostic modalities, treatment and overall survival of mucormycosis in Africa. We searched for relevant publications in PubMed, Google Scholar and African Journal Online databases covering the period 1960-2022. A total of 147 articles were identified, of which 66 were included in the review, detailing 408 individual cases from 12 African countries; 330 (80.9%) from North Africa, 63 (15.4%) from Southern Africa, seven (1.7%) from East Africa, seven (1.7%) from West Africa and a single case (0.2%) from Central Africa. The most frequently described clinical forms were rhino-orbital-cerebral (n = 307, 75.2%) and gastrointestinal (n = 51, 12.5%). Diabetes mellitus, COVID-19, malignancies and neutropaenia were the commonest underlying risks in 203 (49.8%), 101 (24.8%), 65 (15.9%) and 53 (13.0%) cases respectively. Most cases, 296 (72.5%) were diagnosed by histopathology. Fungal aetiology was identified in 38 (9.3%), of which the commonest was Rhizopus oryzae/arrhizus (27/38, 71.1%). Of the 408 cases, 334 (81.9%) patients received antifungal therapy, while 244 (59.8%) had surgery. In cases with a specified outcome, survival rate was 59.1% (228/386). Based on case reporting, a substantial burden of mucormycosis occurs in North Africa but the disease is rarely reported in most of the sub-Saharan region. Establishing a comprehensive registry for standardised data collection could improve understanding of the epidemiology of mucormycosis in the region.

Infección por Aspergillus flavus y Rhizopus oryzae complex en paciente con diabetes mellitus / Infection due to Aspergillus flavus and Rhizopus oryzae complex in a patient with diabetes mellitus

La sinusitis micótica es una condición patológica que puede presentarse en pacientes con diabetes mellitus y estar asociada a una crisis hiperglucémica. Es una entidad agresiva con complicaciones locales que incluyen afectación de la órbita y el sistema nervioso central, y compromiso vascular. A pesar del tratamiento quirúrgico y antimicótico, la mortalidad es de hasta el 75 %. Se describe el caso de una paciente con diagnóstico de cetoacidosis diabética y signos de oftalmoplejía unilateral que llevaron al estudio con resonancia magnética del sistema nervioso central; se encontraron signos de sinusitis, meningitis y cerebritis. Los estudios microbiológicos iniciales fueron negativos, y los biomarcadores galactomanano sérico y el antígeno de Cryptococcus también fueron negativos. Tras el manejo quirúrgico, se llegó a la identificación de Aspergillus flavus y Rhizopus spp. en el tejido de los senos paranasales. La paciente recibió tratamiento con posaconazol y, tras dos meses de seguimiento, había presentado mejoría clínica. La infección fúngica dual y la infección por A. flavus son entidades poco frecuentes y de relevancia clínica, sin casos presentados previamente en nuestro país por lo que este corresponde a un caso de interés clínico.

Fungal sinusitis is a pathology that can occur in patients with diabetes mellitus and be associated with a hyperglycemic crisis. It is an aggressive entity with local complications that include involvement of the orbit or the central nervous system, and vascular involvement. Despite surgical and antifungal treatment, mortality raises up to 75%. We report the case of a female patient with a diagnosis of diabetic ketoacidosis and signs of unilateral ophthalmoplegia, which led to the study with magnetic resonance imaging of the central nervous system, finding signs of sinusitis, meningitis, and cerebritis. Initial microbiological studies were negative, and biomarkers such as serum galactomannan and Cryptococcus antigen were also negative. After surgical management and the identification of Aspergillus flavus and Rhizopus spp. in sinus tissue, the patient received treatment with posaconazole and after two months of follow-up she presented clinical improvement. Dual fungal infection and infection by A. flavus are uncommon and clinically relevant entities, with no cases previously reported in our country, therefore this corresponds to a case of clinical interest.

Microbiology Profile of COVID-19-Associated Rhino-Orbital Mucormycosis Pathogens in South India.

This study describes the microbiological and histopathological features of patients with COVID-19-associated rhino-orbital mucormycosis (ROM) seen at the L V Prasad Eye Institute between May and August 2021. Diagnosed clinically and radiologically, 24 patients with ROM were included in the study. Deep nasal swabs or endoscopically collected nasal swabs or orbital tissues were submitted for microbiological evaluation and in vitro susceptibility testing by microbroth dilution for natamycin, amphotericin B, caspofungin, posaconazole, ketoconazole, and voriconazole. Cultures were processed by 28S ribosomal DNA polymerase chain reaction and molecular sequencing. A portion of orbital tissues was also sent for histopathological evaluation. The age of the patients ranged from 27 to 75 (mean 48.58 ± 14.09) years and the majority (79%) were male. Nineteen patients were known to be diabetic prior to developing ROM and 18 patients had recovered from active COVID-19 infection. Thirteen patients had a history of hospitalization during COVID-19 infection and eight received steroids. Of the 24 samples, microbiological evaluation identified Rhizopus arrhizus in 12, Rhizopus microsporus in 9, Lichtheimia ramosa in 2, and Rhizopus delemar in 1. Twelve isolates were tested for antifungal susceptibility and all were susceptible to natamycin and amphotericin B. The susceptibility to posaconazole was high, with minimum inhibitory concentration (MIC) < 2 µg/mL for 10/12 (84%) isolates, whereas the MIC of other drugs varied. Histopathological examination of tissues showed acute fulminant disease, granuloma formation, and vascular invasion by the fungal pathogens in these specimens. Rhizopus arrhizus was predominantly associated with ROM and most isolates were susceptible to amphotericin B and posaconazole. Further studies are needed to corroborate the findings and explain possible underlying links.

Screening of potential antigens from whole proteome and development of multi-epitope vaccine against Rhizopus delemar using immunoinformatics approaches.

Mucormycosis is a deadly fungal disease mainly caused by Rhizopus oryzae (strain 99-880), also known as Rhizopus delemar. Previously, mucormycosis occurs in immunocompromised patients of diabetes mellitus, cancer, organ transplant, etc. But there was a drastic increase in mucormycosis cases in the ongoing COVID-19 pandemic. Despite several available therapies and antifungal treatments, the mortality rate of mucormycosis is about more than 50%. Currently, there is no vaccine available in the market for mucormycosis that urgently needs to develop a potential vaccine against mucormycosis with high efficacy. In the present study, we have screened 4 genome-derived predicted antigens (GDPA) through sequential filtration of the whole proteome of R. delemar using different benchmarked bioinformatics tools. These 4 GDPA along with 4 randomly selected experimentally reported antigens (ERA) were sourced for prediction of B- and T- cell epitopes and utilized in designing of two potential multi-epitope vaccine candidates which can induce both innate and adaptive immunity against R. delemar. Besides these, comparative immune simulation studies and in silico cloning were performed using L. lactis as an expression system for their possible uses as oral vaccines. This is the first multi-epitope vaccine designed against R. delemar through systematic pipelined reverse vaccinology and immunoinformatic approaches. Although the wet-lab based experimental validation of designed vaccines is required before testing in the preclinical model, the current study will significantly help in reducing the cost of experimentation as well as improving the efficacy of vaccine therapy against mucormycosis and other pathogenic diseases.Communicated by Ramaswamy H. Sarma.

Rhizopus oryzae fermentation wastewater nutrient removal coupling protein fodder production by employing Chlorella pyrenoidosa.

The scenario was to investigate feasibilities of employing Chlorella pyrenoidosa for Rhizopus oryzae fermentation wastewater nutrient removal coupling protein fodder production. Results stated that TN, TP, NH3-N, COD, BOD removal reached 99.79%, 94.70%, 98.80%, 97.60%, 99.60% to acquire discharge permit under fed-batch manipulation, whilst the peaked protein yield (19.94 g/L) was 6.04-fold more than batch manipulation. Rhizopus oryzae fermentation wastewater feeding C. pyrenoidosa was praised as high-quality protein not only with 26.78% essential amino acids and essential amino acids/nonessential amino acids value of 0.84 but also pathogenic bacteria and heavy metal loads complying with fodder standards. In vitro digestibility of dry matter, protein, lipid, and starch achieving 80.07%, 92.13%, 95.93%, 91.9% and bioavailability of polypeptides, triglycerides, free fatty acids, and oligosaccharides displaying 98.67%, 87.12%, 93.86%, 30.21%, which were roughly-equivalent to corn/soybean fodder. The findings formed exemplifications in utilizing other microalgal systems for wastewater nutrient removal coupling chemicals production.

COVID-19-associated-mucormycosis: possible role of free iron uptake and immunosuppression.

COVID-19-associated-mucormycosis, commonly referred to as the "Black Fungus," is a rare secondary fungal infection in COVID-19 patients prompted by a group of mucor molds. Association of this rare fungal infection with SARS-CoV-2 infection has been declared as an endemic in India, with minor cases in several other countries around the globe. Although the fungal infection is not contagious like the viral infection, the causative fungal agent is omnipresent. Infection displays an overall mortality rate of around 50%, with many other secondary side effects posing a potential threat in exacerbating COVID-19 mortality rates. In this review, we have accessed the role of free iron availability in COVID-19 patients that might correlate to the pathogenesis of the causative fungal agent. Besides, we have analyzed the negative consequences of using immunosuppressive drugs in encouraging this opportunistic fungal infection.

Invasive Mucormycosis in Children With Malignancies: Report From the Infection Working Group of the Hellenic Society of Pediatric Hematology-Oncology.

Mucormycosis is an invasive, life-threatening fungal infection that mainly affects immunocompromised hosts. We collected data of pediatric mucormycosis cases from all 7 Greek Hematology-Oncology Departments for the years 2008-2017. Six cases of invasive mucormycosis diagnosed during treatment for malignancies were included in the study. In 4 children (66%) mucormycosis occurred within the first 20 days after diagnosis of the underlying disease. Two cases were classified as proven mucormycosis and 4 as probable. The most frequently recorded species was Rhizopus arrhizus (2 patients), followed by Mucor spp (1), and Lichtheimia spp (1). All patients received liposomal amphotericin B. Combined antifungal treatment was used in 5 cases. Surgical excision was performed in 4 cases (66%). Two patients died at 6 and 12 months after the diagnosis, respectively, 1 (17%) because of mucormycosis. Our data suggest that mucormycosis may occur early after the initiation of intensive chemotherapy in children with malignancies.

Metaproteomic investigation of functional insight into special defined microbial starter on production of fermented rice with melanogenesis inhibition activity.

Fermentation of rice grains requires diverse metabolic enzymes to be synchronously synthesized by the microbial community. Although many studies have used a metaproteomic approach to investigate the roles of microorganisms in improving the flavor of fermented foods, their roles in producing compounds with biological activity have not yet been reported. In a previous study the ferment obtained from unpolished black rice (UBR) fermented with a defined microbial starter (De-E11), comprised of Rhizopus oryzae, Saccharomycopsis fibuligera, Saccharomyces cerevisiae, and Pediococcus pentosaceus, (fermented UBR; FUBR) showed a strong melanogenesis inhibition activity in B16F10 melanoma cells. Hence, in this study, the roles of these microorganisms in producing the melanogenesis inhibitor(s) in FUBR was investigated using a metaproteomic approach. The melanogenesis inhibition activity of the FUBR liquid (FR-Liq) was found to increase with longer fermentation times. R. oryzae and S. cerevisiae were the major hosts of proteins related to the biosynthesis of melanogenesis inhibitor(s) in the FUBR. During fermentation, the enzymes involved in the degradation of UBR and in the carbohydrate metabolic process were identified. These enzymes were associated with the process of releasing of bioactive compound(s) from UBR and the synthesis of organic acids from the microorganisms, respectively. In addition, enzymes involved in the synthesis of some known melanogenesis inhibitor(s) and in the degradation of the melanogenesis stimulator (arsenate) were detected. Varying the combination of microorganisms in the De-E11 starter to produce the FR-Liq revealed that all four microorganisms were required to produce the most potent melanogenesis inhibition activity. Taken together with the metaproteomics results, this suggested that the microorganisms in De-E11 synchronously synthesize the FR-Liq with melanogenesis inhibition activity. In conclusion, this information on the metaproteome in FUBR will increase our understanding of the microbial metabolic modes and could lead to knowledge-based improvements in the fermented rice process to produce melanogenesis inhibitor(s).

A 71-Year-Old Man With Chest Pain and a Solitary Pulmonary Mass.

CASE PRESENTATION: A 71-year-old man was admitted to our hospital because of diffuse chest pain and a mass on routine chest radiography. He did not report cough, dyspnea, fever, night sweats, or weight loss. His medical history was remarkable for chronic lymphocytic leukemia diagnosed 13 years before presentation, and secondary myelodysplastic syndrome diagnosed 2 years before the onset of the current symptoms. As a curative approach, he had received a matched unrelated stem cell transplantation 16 months earlier, and he had been in complete remission since. He developed chronic graft-vs-host disease, presenting mainly as oral ulceration (grade 1, according to National Institute of Health consensus criteria), which had been treated with oral cyclosporine and extracorporeal photopheresis. The immunosuppression had been tapered 6 months before presentation. Routine medication included co-trimoxazole prophylaxis twice per week. He had no known allergies, and he denied recent travels and sick contacts.

Evaluation of biochemical and antioxidant dynamics during the co-fermentation of dehusked barley with Rhizopus oryzae and Lactobacillus plantarum.

Barley is an ancient and important functional crop. In this study, the biochemical and antioxidant dynamics of dehusked barley during co-fermentation with Lactobacillus plantarum and Rhizopus oryzae were investigated. Results showed that amino acid nitrogen, soluble protein, <10 kDa peptide, and free phenolic contents increased with fermentation time; whereas the lactic acid bacteria count, reducing sugar, and free flavonoid contents showed a tendency to increase first and then decrease. SDS-PAGE analysis indicated that bands at >25 and <18.4 kDa were shown with improved intensity with time, whereas bands at 18.4-25 kDa were disappeared. Additionally, fermentation time led to an increase in DPPH, hydroxyl, ABTS+ radical scavenging activity, and ferric reducing antioxidant power. Thus, this study demonstrated that co-fermentation with L. plantarum and R. oryzae could improve nutrition and potential bioactivity of barley, and use barley as a good solid-state food carrier for probiotics. PRACTICAL APPLICATIONS: Barley is rich in nutrition and has a huge production. However, due to its high-fiber and special protein composition, there is a certain palatability problem whether it is used to produce flour or other products. Therefore, most of the barley is destined to animal feed and malt, and only a small part is used directly for human consumption. Aiming at this problem, on the basis of the optimization of the fermentation process at early stage, our results further demonstrated co-fermentation with L. plantarum and R. oryzae could enhance the nutritional value and potential bioactivity of barley, thus providing a novel approach to develop functional barley food and improve the direct utilization rate of barley in food processing.

Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.