A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

Mucormycosis in Liver Allograft Following Transplant for Secondary Biliary Cirrhosis.

Mucormycosis, a group of opportunistic mycoses caused by Mucorales, present a significant threat to immunocompromised patients. In this report, we present the case of a 57-year-old male patient who underwent liver transplant for secondary biliary cirrhosis following inadvertent bile duct injury. Despite initial satisfactory postoperative evolution, the patient developed fever, and imaging revealed a suspicious lesion. Preliminary culture growth suggested a filamentous fungus, leading to initiation of liposomal amphotericin B. However, the lesion progressed, and a surgical debridement was necessary. During surgery, involvement of the liver dome and diaphragm was observed, and a nonanatomical hepatectomy was performed. Despite efforts, the patient's condition deteriorated, ultimately resulting in multiple organ failure and mortality. This case emphasizes the challenging nature of mucormycosis in livertransplant recipients.

Central Retinal Artery Occlusion in Rhino-Orbital-Cerebral Mucormycosis: An Inflammatory-Prothrombotic State.

PURPOSE: The aim was to evaluate patient profiles of rhino-orbital-cerebral mucormycosis (ROCM) cases with central retinal artery occlusion (CRAO) postcoronavirus disease 2019. DESIGN: A nonrandomized retrospective case-control study. METHODS: The ROCM cases presenting with CRAO were compared with a control ROCM group without CRAO at a tertiary care center. Demography, systemic status, clinical features, histopathology, imaging, and blood profile were assessed for any specific risk factors. RESULTS: A total of 12 patients were seen in the CRAO group and 16 in the non-CRAO group. The male-to-female ratio was 3:1 with a mean age of 49.5 years. In the CRAO group, 75% had diabetes mellitus with mean hemoglobin A1c of 9.03%, and 66.7% had received steroid treatment. All cases were histopathologically confirmed positive for mucor. There was a significant difference in mean D-dimer and serum ferritin between the 2 groups, with higher level in the CRAO group. All patients with CRAO had light perception-negative vision, with total ophthalmoplegia and proptosis seen in 66.7% of cases. Four patients had orbital apex involvement, 5 had cavernous sinus involvement, and 8 had intracranial involvement in the CRAO group. CONCLUSIONS: Inflammatory markers D-dimer and serum ferritin were significantly associated with CRAO, suggestive of hyperinflammatory and hypercoagulable state. A high index of suspicion should be maintained in cases with elevated markers and prophylactic anticoagulants can be started to prevent CRAO in a subset of patients.

Resolving the equation between mucormycosis and COVID-19 disease.

The COVID-19 patients, both infected and recovered are rapidly contracting mucormycetes infections due to the 'Mucorales' order, under Zygomycetes class of fungi. The mucorales fungi commonly known to exist in our natural surroundings including soil, but the frequency of incidences was never rampant. This sudden spike in infections, is locally known as 'black fungus,' and is affecting various organs, including- eyes, sinuses, nose, brain, skin, intestine, lungs, etc. The severity of situation is ascertainable from the fact that, in certain cases surgical eye/jaws removal persists as the only viable option to avert mortality, as therapeutic interventions are limited. This epidemic situation intrigued experts to investigate the probable reason behind this unpredicted escalation in reported cases, including in recuperated COVID-19 patients, as person-to-person spread of infection is not common. The comparison of physiological parameters in healthy and COVID-19 afflicted patients highlights that the underlying conditions including diabetes mellitus, steroidal therapy, lymphopenia (decreased CD4+ and CD8+ lymphocytes), deregulated cytokine release storm, elevated free iron levels (hemosiderosis) in blood and insulin insensitivity are playing major roles in deteriorating conditions in rarely pathogenic fungal infections. This review is an attempt to explain the rationalities that makes people vulnerable to mucormycetes infection.

COVID associated mucormycosis: A preliminary study from a dedicated COVID Hospital in Delhi.

BACKGROUND: It is an incontrovertible fact that the Rhino Orbital Cerebral Mucormycosis (ROCM) upsurge is being seen in the context of COVID-19 in India. Briefly presented is evidence that in patients with uncontrolled diabetes, a dysfunctional immune system due to SARS-COV-2 and injudicious use of corticosteroids may be largely responsible for this malady. OBJECTIVE: To find the possible impact of COVID 19 infection and various co-morbidities on occurrence of ROCM and demonstrate the outcome based on medical and surgical interventions. METHODOLOGY: Prospective longitudinal study included patients diagnosed with acute invasive fungal rhinosinusitis after a recent COVID-19 infection. Diagnostic nasal endoscopy (DNE) was performed on each patient and swabs were taken and sent for fungal KOH staining and microscopy. Medical management included Injection Liposomal Amphotericin B, Posaconazole and Voriconazole. Surgical treatment was restricted to patients with RT PCR negative results for COVID-19. Endoscopic, open, and combined approaches were utilized to eradicate infection. Follow-up for survived patients was maintained regularly for the first postoperative month. RESULTS: Out of total 131 patients, 111 patients had prior history of SARS COVID 19 infection, confirmed with a positive RT-PCR report and the rest 20 patients had no such history. Steroids were received as a part of treatment in 67 patients infected with COVID 19. Among 131 patients, 124 recovered, 1 worsened and 6 died. Out of 101 known diabetics, 98 recovered and 3 had fatal outcomes. 7 patients with previous history of COVID infection did not have any evidence of Diabetes mellitus, steroid intake or any other comorbidity. CONCLUSION: It can be concluded that ROCM upsurge seen in the context of COVID-19 in India was mainly seen in patients with uncontrolled diabetes, a dysfunctional immune system due to SARS-COV-2 infection and injudicious use of corticosteroids.

Emerging fungal infections: focus on Saksenaea Erythrospora.

INTRODUCTION: In recent times, improved diagnostic techniques have revealed an alarming number of cases of mucormycosis in immunocompetent individuals. The Saksenaea species, is a rare cause of mucormycosis, and is often associated with skin and subcutaneous infection due to trauma in both immunocompromised and immunocompetent subjects. The purpose of this study was therefore, through a review of the literature, to investigate the problem of infections caused by Saksenaea Erythrospora, evaluating the clinical manifestations of the infection, the triggering factors, the therapies and patients' outcomes, implementing and updating what already reported in literature. METHODS: A research of peer-reviewed literature in the electronic databases MEDLINE (PubMed) and Scopus was conducted in the period June 2020-January 2021 using the key word "Saksenaea erythrospora". Studies in Italian, English, French, Spanish focused on cases of Saksenaea erythrospora were included, without time restrictions. Studies that provided ambiguous or insufficient data were excluded. RESULTS: Bibliographic research yielded 23 publications; 7 were included in the review. The studies were published between 2011 and 2015 and involved a total of 11 patients of average age 37.9 years (SD 17.23) hospitalized in several hospitals in: USA, India, Argentina, Colombia, Thailand. 6 patients were women, 5 men. All patients had an almost normal immune status. The causes of the infection were: injections, traumas, surgery. Two patients, despite surgical and medical therapy, died. CONCLUSIONS: Our review partially updated what already published, because only one new study was found. Serious necrotizing infections from Saksenaea erythrospora have been observed in recent years and a early identification and timely management are essential to reduce morbidity and mortality. A greater awareness and education about the risks deriving from carrying out surgical procedures abroad, especially in precarious hygiene situations, could be additional effective weapons to reduce the incidence of these infections.

Rhino-orbital-cerebral-mucormycosis in COVID-19: A systematic review.

Since the onset of COVID-19 pandemic, parallel opportunistic infections have also been emerging as another disease spectrum. Among all these opportunistic infection, mucormycosis has become a matter of concern with its rapid increase of cases with rapid spread as compared to pre-COVID-19 era. Cases have been reported in post-COVID-19-related immune suppression along with the presence of comorbidity which adds on the deadly outcome. There is no systematic review addressing the issue of COVID-19-associated mucormycosis. This is the first systematic review of published studies of mucormycosis associated with COVID-19. The aim was to analyze the real scenario of the disease statement including all the published studies from first November 2019 to 30th June to analyze the contemporary epidemiology, clinical manifestations, risk factor, prognosis, and treatment outcome of COVID-19 associated rhino-orbito-cerebral-mucormycosis. A comprehensive literature search was done in following databases, namely, PubMed, Google Scholar, Scopus, and EMBASE using keywords mucormycosis, rhino orbital cerebral mucormycosis, COVID-19, and SARS-CoV-2 (from November 01, 2019 to June 30, 2021). Our study shows that, while corticosteroids have proved to be lifesaving in severe to critical COVID-19 patients, its indiscriminate use has come with its price of rhino-orbito-cerebral mucormycosis epidemic, especially in India especially in patients with preexisting diabetes mellitus with higher mortality. Corticosteroid use should be monitored and all COVID-19 patients should be closely evaluated/monitored for sequelae of immunosuppression following treatment.

A Comparative Analysis of Mucormycosis in Immunosuppressed Hosts Including Patients with Uncontrolled Diabetes in the Southwest United States.

BACKGROUND: Mucormycosis (zygomycosis) is an invasive fungal infection that carries a high risk of morbidity and mortality. Uncontrolled diabetes mellitus and other immunocompromising conditions are risk factors for mucormycosis development. We here describe the differences in characteristics and outcomes of mucormycosis among solid organ transplant, hematological malignancy, and diabetes mellitus groups at our institution. METHODS: We conducted a retrospective chart review over the period of 2009-2020, with identifying patients using the International Classification of Diseases, Ninth and Tenth Revisions. Clinical, laboratory, and outcome data were collected. RESULTS: There were 28 patients identified: 7 solid organ transplant, 3 hematological malignancy, and 18 diabetes mellitus patients were included in the study. Three solid organ transplant patients experienced an episode of rejection, and another 3 had cytomegalovirus infection prior to presenting with mucormycosis. Four of seven solid organ transplant patients had a history of diabetes mellitus, but the median hemoglobin A1C was lower than in the diabetes mellitus group (6.3 vs 11.5; P = .006). The mortality rate difference between solid organ transplant and diabetes mellitus was not statistically significant: 2/7 (28.57%) vs 5/18 (27.78%); P = .66. Patients with bilateral disease (pulmonary or sinus) had significantly higher mortality (80% vs 13%, P = .008). There was no difference in mortality outcomes among the different types of antifungal therapies administered. CONCLUSION: A multispecialty approach is imperative in mucormycosis therapy. While the underlying risk factors were different, the outcomes were comparable for the solid organ transplant and diabetes mellitus groups. Future larger and longitudinal studies are recommended.

Rhinocerebral mucormycosis secondary to severe acute pancreatitis and diabetic ketoacidosis: a case report.

INTRODUCTION: Rhinocerebral mucormycosis is a rare and severe form of opportunistic fungal infection that can develop rapidly and cause significant mortality, particularly among diabetic patients suffering from ketoacidosis. Diagnosing rhinocerebral mucormycosis during the early stages of infection is challenging. CASE PRESENTATION: We describe a case of rhinocerebral mucormycosis secondary to severe acute pancreatitis in a patient suffering from diabetic ketoacidosis. In this case, the condition was not diagnosed during the optimal treatment window. we therefore provide a thorough overview of related clinical findings and histopathological characteristics, and we discuss potential differential diagnoses. CONCLUSIONS: In summary, we described a case of rhinocerebral mucormycosis secondary to severe acute pancreatitis in a patient suffering from diabetic ketoacidosis, with the optimal treatment window for this condition having been missed. This report suggests that a definitive mucormycosis diagnosis can be made based upon tissue biopsy that reveals the presence of characteristic hyphae. Early diagnosis and treatment are essential in order to improve patient prognosis.

In Vivo and In Vitro Impairments in T Helper Cell and Neutrophil Responses against Mucor irregularis in Card9 Knockout Mice.

Mucor irregularis is a frequently found fungus in Asia, especially China, and it causes primary cutaneous mucormycosis with a high rate of disfigurement. Caspase recruitment domain-containing protein 9 (Card9) is an essential adaptor molecule downstream of C-type lectin receptors. It mediates the activation of nuclear factor kappa B (NF-κB), regulates T helper 1 (Th1) and Th17 differentiation, and plays an important role in fungal immune surveillance. CARD9 deficiency correlates with the increased susceptibility to many fungal infections, including cutaneous mucormycosis caused by M. irregularis However, the underlying immunological mechanisms were not elucidated. Our study established a murine model of subcutaneous M. irregularis infection, and we isolated immune cells, including bone marrow-derived macrophages, bone marrow-derived dendritic cells, naive T cells, and neutrophils, from wild-type (WT) and Card9 knockout (Card9-/- ) mice to examine the antifungal effect of Card9 on M. irregularis in vivo and in vitroCard9-/- mice exhibited increased susceptibility to M. irregularis infection. Impaired local cytokine and chemokine production, NF-κB (p65) activation, and Th1/17 cell differentiation and partially impaired neutrophil-dependent antifungal immunity were observed in Card9-/- mice. This work enriches our knowledge of the relationship between CARD9 deficiency and mucormycosis.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

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.

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.

Diagnosis of oesophageal mucormycosis managed with medical therapy alone.

Mucormycosis is an invasive mould that can cause aggressive infection, particularly in immunocompromised patients. Though oesophageal mucormycosis is relatively rare, it remains an elusive and devastating manifestation of this disease. The management is also challenging, due to surgical morbidity and contraindications such as thrombocytopenia in immunocompromised hosts. In this report, we present the case of a 60-year-old Lebanese man with newly diagnosed acute myeloid leukaemia who developed oesophageal mucormycosis after induction chemotherapy with idarubicin/cytarabine (7+3). The diagnosis was made when the patient developed febrile neutropenia and odynophagia. CT scan of the chest revealed a thickened oesophagus. Oesophagogastroduodenoscopy with biopsy, histopathology and PCR were performed, resulting in the diagnosis of Rhizopus microsporus The patient was successfully treated with liposomal amphotericin B and salvage posaconazole therapy without surgical intervention. We reviewed the clinical characteristics of the six published oesophageal mucormycosis reports from the literature.

Identification of Mucor circinelloides antigens recognized by sera from immunocompromised infected mice / Identificación de antígenos de Mucor circinelloides reconocidos por sueros de ratones inmunodeprimidos infectados

BACKGROUND: Mucor circinelloides is an opportunistic fungus capable of causing mucormycosis, a highly aggressive infection of quick spreading. Besides, it also has a high mortality rate due to late diagnosis and difficult treatment. AIMS: In this study we have identified the most immunoreactive proteins of the secretome and the total protein extract of M. circinelloides using sera from immunocompromised infected mice. METHODS: The proteins of the secretome and the total extract were analyzed by two-dimensional electrophoresis and the most immunoreactive antigens were detected by Western Blot, facing the sera of immunocompromised infected mice to the proteins obtained in both extracts of M. circinelloides. RESULTS: Seven antigens were detected in the secretome extract, and two in the total extract, all of them corresponding only to three proteins. The enzyme enolase was detected in both extracts, while triosephosphate isomerase was detected in the secretome, and heat shock protein HSS1 in the total extract. CONCLUSIONS: In this work the most immunoreactive antigens of the secretome and the total extract of M. circinelloides were identified. The identified proteins are well known fungal antigens and, therefore, these findings can be useful for future research into alternatives for the diagnosis and treatment of mucormycosis

ANTECEDENTES: Mucor circinelloides es un hongo oportunista causante de la mucormicosis, una infección altamente agresiva y de rápida expansión. Además, también presenta una alta mortalidad debido al diagnóstico tardío y el difícil tratamiento. OBJETIVOS: En este estudio se han identificado las proteínas más inmunorreactivas del secretoma y del extracto total de proteínas de M. circinelloides mediante el uso de sueros obtenidos de ratones inmunodeprimidos infectados. MÉTODOS: Las proteínas del secretoma y del extracto total se analizaron mediante electroforesis bidimensional y se detectaron los antígenos más inmunorreactivos mediante Western Blot, enfrentando el suero de los ratones inmunodeprimidos infectados a las proteínas obtenidas en ambos extractos de M. circinelloides. RESULTADOS: Se identificaron 7 antígenos en el secretoma y 2 en el extracto total, todos ellos correspondientes a 3 proteínas. La enolasa se detectó en ambos extractos, mientras que la triosafosfato isomerasa se detectó en el secretoma, y la proteína de choque térmico HSS1 en el extracto total. CONCLUSIONES: En este trabajo se identificaron los antígenos más inmunorreactivos del secretoma y del extracto total de M. circinelloides. Todas las proteínas identificadas son antígenos fúngicos muy conocidos y, por ello, estos resultados pueden ser de gran utilidad en futuras investigaciones relacionadas con la mejora del diagnóstico y el tratamiento de la mucormicosis