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.

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.

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.

Chronic invasive fungal rhinosinusitis vs sinonasal squamous cell carcinoma: the differentiating value of MRI.

OBJECTIVES: To investigate MRI features in discriminating chronic invasive fungal rhinosinusitis (CIFRS) from sinonasal squamous cell carcinomas (SNSCC). METHODS: MRI findings of 33 patients with CIFRS and 47 patients with SNSCC were retrospectively reviewed and compared. Multivariate logistic regression analysis was performed to identify significant imaging features in distinguishing between CIFRS and SNSCC. The ROC curves and the AUC were used to evaluate diagnostic performance. RESULTS: There were significant differences in cavernous sinus involvement (p < 0.001), sphenoid sinus involvement (p < 0.001), meningeal involvement (p = 0.024), T2 signal intensity (p = 0.006), and enhancement pattern (p < 0.001) between CIFRS and SNSCC. Multivariate logistic regression analysis identified cavernous sinus involvement (odds ratio [OR] = 0.06, 95% confidence interval [95% CI] = 0.02-0.20) and sphenoid sinus involvement (OR = 0.14, 95% CI = 0.05-0.45) as significant indicators for CIFRS and T2 isointensity to gray matter (OR = 4.44, 95% CI = 1.22-16.22) was a significant indicator for SNSCC. ROC curve analysis showed the AUC from a combination of three imaging features was 0.95 in differentiating CIFRS and SNSCC. CONCLUSIONS: MRI showed significant differences between CIFRS and SNSCC features. In immunocompromised patients, a sinonasal hypointense mass on T2WI with septal enhancement or loss of contrast enhancement, and involvement of cavernous sinus, sphenoid sinus, and meninges strongly suggest CIFRS. KEY POINTS: • Chronic invasive fungal rhinosinusitis (CIFRS) is often difficult to distinguish from sinonasal squamous cell carcinomas (SNSCC) in clinical practice. • Cavernous sinus and sphenoid sinus involvement appear to be significant indicators for CIFRS. T2 isointensity to gray matter appears to be a significant indicator for SNSCC. • Loss of contrast enhancement and septal enhancement can be used to distinguish CIFRS from SNSCC with a high degree of specificity.

Pulmonary Mucormycosis: Risk Factors, Radiologic Findings, and Pathologic Correlation.

Pulmonary mucormycosis (PM) is an uncommon fungal infection most often seen in immunocompromised patients. The fungus grows on decaying food, soil, and animal excrement. Patients usually become infected by inhalation of spores. The most common risk factors include diabetes mellitus, hematologic malignancy, and solid organ or stem cell transplant. PM can have a nonspecific appearance at imaging. For example, early imaging may show peribronchial ground-glass opacity. Later, the disease progresses to consolidation, nodules, or masses. Because patients are usually immunocompromised, the differential diagnosis often includes invasive pulmonary aspergillosis (IPA). Various radiologic findings suggestive of PM have been identified to help differentiate it from IPA. For example, the reverse halo sign is more closely associated with PM than with IPA. The reverse halo sign is an area of ground-glass opacity surrounded by a rim of consolidation. In addition, the presence of pleural effusions and more than 10 nodules is more suggestive of PM than it is of IPA. PM can progress rapidly in neutropenic patients. Identification of the hyphae in tissue by using endobronchial or percutaneous sampling can allow differentiation from IPA and help confirm the diagnosis of mucormycosis. Because of the high mortality rate associated with PM, early identification of the disease is critical for an improved likelihood of survival. A multimodality treatment approach with antifungal agents and surgical débridement has been shown to improve outcomes. The authors review the risk factors for PM, describe its imaging appearance and disease process, and describe the treatment of the disease. ©RSNA, 2020.

The geographical region of origin determines the phagocytic vulnerability of Lichtheimia strains.

Mucormycoses are life-threatening infections that affect patients suffering from immune deficiencies. We performed phagocytosis assays confronting various strains of Lichtheimia species with alveolar macrophages, which form the first line of defence of the innate immune system. To investigate 17 strains from four different continents in a comparative fashion, transmitted light and confocal fluorescence microscopy was applied in combination with automated image analysis. This interdisciplinary approach enabled the objective and quantitative processing of the big volume of image data. Applying machine-learning supported methods, a spontaneous clustering of the strains was revealed in the space of phagocytic measures. This clustering was not driven by measures of fungal morphology but rather by the geographical origin of the fungal strains. Our study illustrates the crucial contribution of machine-learning supported automated image analysis to the qualitative discovery and quantitative comparison of major factors affecting host-pathogen interactions. We found that the phagocytic vulnerability of Lichtheimia species depends on their geographical origin, where strains within each geographic region behaved similarly, but strongly differed amongst the regions. Based on this clustering, we were able to also classify clinical isolates with regard to their potential geographical origin.

Impairment of Th cell response in Card9 knockout mice with cutaneous mucormycosis caused by Rhizopus arrhizus.

Card9 is a signalling adaptor protein in the downstream of many innate pattern recognition receptors (PRRs) and exerts a significant role in antifungal immunity. To date, Card9 deficiency has been reported to be related to increased susceptibility to many fungal infections. In this study, we established mucormycosis murine model of Rhizopus arrhizus (R. arrhizus) using wild-type (WT) mice and Card9 knockout (Card9-/- ) mice to investigate the antifungal effect of Card9 against R. arrhizus infection. Card9-/- mice were more susceptible to R. arrhizus infection than WT mice, which could be related to the impaired NF-κB pathway activation, local cytokine production and Th cell responses in Card9-/- mice.

Early necrotic skin lesions after a ABO-incompatible kidney transplantation: The threat of Cunninghamella Spp.

A 49-year-old man underwent ABO-incompatible kidney transplantation with a living donor. At day 33 post-transplantation, he presented with undiagnosed epilepsy with generalized tonic-clonic seizures. At day 44 post-transplantation, he developed left-sided pneumonia attributed to Aspergillus fumigatus and treatment with liposomal amphotericin B was initiated. At day 51 post-transplantation, necrotic skin lesions appeared. DNA sequencing in a fresh cutaneous biopsy finally identified Cunninghamella Spp., a member of the order Mucorales. Unfortunately, the necrotic lesions spread, and the patient died at day 60 post-transplantation. This case report highlights the infectious risk related to ABO-incompatible kidney transplantation and suggests a requirement for rapid identification of every skin lesion, even in the early phases of immunosuppression.

Analysis of the Clinical Features of Tracheobronchial Fungal Infections with Tumor-Like Lesions.

BACKGROUND: Tracheobronchial fungal infections (TBFI) cause life-threatening complications in immunocompromised hosts but are rarely reported. Misdiagnosis and delayed antifungal treatment are associated with the high mortality rate of patients with TBFI. OBJECTIVES: This study analyzed the bronchoscopic features of TBFI and their roles in the early diagnosis of TBFI. METHODS: The demographic, clinical, radiologic, and bronchoscopic data of 53 patients diagnosed with TBFI in our department during a 15-year period were retrospectively analyzed. RESULTS: Most of the TBFI patients were male, and mass was the most common radiologic abnormality. Obvious predilection in primary bronchus distributions was observed. 41.9% of the 43 Aspergillus tracheobronchitis (AT) patients, 70% of the 10 tracheobronchial mucormycosis (TM) patients, and 100% of the 3 endobronchial cryptococcosis patients had been misdiagnosed as having cancer on bronchoscopy because of the presence of tumor-like lesions. The most common features of AT were bronchial occlusion with a mass or mucosal necrosis, bronchial stenosis with mucosal hyperplasia, or uneven mucosa. The main descriptions of TM were bronchial stenosis or obstruction due to mucosal necrosis, uneven mucosa, or a mass. The endoscopic characteristics of endobronchial cryptococcosis included occlusion due to uneven mucosa or mass, or external compressive stricture. CONCLUSION: Immunocompromised patients and immunocompetent patients with underlying disease displaying tumor-like lesions on bronchoscopy should be differentially diagnosed with cancer. Bronchial biopsy is indispensable for the early diagnosis of TBFI.

Isolated Pulmonary Mucormycosis in an immunocompetent patient.

Mucormycosis is a life-threatening fungal infection that occurs mainly in immunocompromised patients. Its occurrence isolated in the lung rare and carries a high mortality risk if untreated. We report the case of a 76-year old male immunocompetent patient, under treatment for pulmonary tuberculosis, admitted to the emergency department with hemoptysis. Bronchoscopy was performed and active bleeding from the middle lobe bronchus was found. Chest CT scan identified a solitary cavitary lesion in the middle lobe. The patient was proposed for urgent open middle lobectomy. Postoperative period was uneventful. Pulmonary mucormycosis was confirmed and adjuvant therapy with Amphotericin B was performed for 30 days. Despite its rarity, mucormycosis prevalence is expected to raise together with increasing number of immunocompromised patients. A high level of suspicion is recommended as early diagnosis can be determinant.

Pulmonary mucormycosis: serial morphologic changes on computed tomography correlate with clinical and pathologic findings.

PURPOSE: To evaluate serial computed tomography (CT) findings of pulmonary mucormycosis correlated with peripheral blood absolute neutrophil count (ANC). MATERIALS AND METHODS: Between February 1997 and June 2016, 20 immunocompromised patients (10 males, 10 females; mean age, 48.9 years) were histopathologically diagnosed as pulmonary mucormycosis. On initial (n=20) and follow-up (n=15) CT scans, the patterns of lung abnormalities and their changing features on follow-up scans were evaluated, and the pattern changes were correlated with ANC changes. RESULTS: All patients were immunocompromised. On initial CT scans, nodule (≤3cm)/mass (>3cm) or consolidation with surrounding ground-glass opacity halo (18/20, 90%)) was the most common pattern. On follow-up CT, morphologic changes (13/15, 87%) could be seen and they included reversed halo (RH) sign, central necrosis, and air-crescent sign. Although all cases did not demonstrate the regular morphologic changes at the same timeline, various combinations of pattern change could be seen in all patients. Sequential morphologic changes were related with recovering of ANC in 13 of 15 patients. CONCLUSION: Pulmonary mucormycosis most frequently presents as consolidation or nodule/mass with halo sign at CT. Morphologic changes into RH sign, central necrotic cavity or air-crescent sign occur with treatment and recovery of ANC. KEY POINTS: • Pulmonary mucormycosis showed various CT-morphology including CT halo sign • Pulmonary mucormycosis had trends of serial morphologic changes on follow-ups • Recovery of absolute neutrophil count changed CT-morphology of mucormycosis in immune-compromised patients.

Successful treatment of invasive mucormycosis with isavuconazole in pediatric patients.

BACKGROUND: Invasive mold infections (IMIs) are a leading cause of mortality among immunocompromised patients. Isavuconazole is a new drug that shows promise in the adult population for the treatment of IMIs. No data regarding the use of isavuconazole in pediatric patients have been published. METHODS: Patients with a diagnosis of IMI from our pediatric hemato-oncology division, treated with isavuconazole between 2010 and 2016, were identified using the hospital's computerized database. Data including demographics, clinical course, and outcome were collected. Pharmacokinetic samples were obtained from two younger patients to guide dosing. RESULTS: In total, three patients (4.5, 5, and 19 years of age) with invasive mucormycosis who were treated with isavuconazole were identified. All patients were treated with isavuconazole as a second line therapy and experienced improvement following the initiation of this treatment. CONCLUSIONS: Based on our limited clinical experience, isavuconazole may be a safe and effective treatment option for children and adolescents afflicted by IMI. Prospective clinical trials should be performed in order to evaluate the pharmakokinetics and safety of isavuconazole in the pediatric population.

Successful treatment of pulmonary mucormycosis in two pediatric hematopoietic stem cell transplant patients.

Pulmonary mucormycosis diagnosed immediately after hematopoietic stem cell transplantation frequently portends a poor prognosis. However, here we describe two cases in children that were treated successfully to highlight the efficacy of a multidisciplinary approach. Despite diagnosis in the immediate post-transplant period and requirement for ongoing immunosuppression to prevent or treat GVHD, both are long-term survivors due to early surgical debridement with transfusion support and prompt initiation of targeted antifungal therapy. In the absence of evidence-based treatment guidelines, survival of pulmonary mucormycosis is achievable even in high-risk patients with a multidisciplinary team to guide management.

Intra-arterial spread of Mucormycetes mediates early ischemic necrosis of brain and suggests new venues for prophylactic therapy.

Intracranial invasion by Mucormycosis carries high mortality mostly related to arterial occlusion and ischemic necrosis. We report clinical, imaging and autopsy findings in an adult immunodeficiency syndrome (AIDS) patient with fungal infection extending from a tooth. We report a striking discordance between a restriction of fungal growth to the initial branches of the circle of Willis and extensive ischemic infarcts of deep brain structures. This lends to a suggestion of apparently lost opportunities for brain salvage and prompts a re-assessment of clinical approaches to treat mucormycosis.