Pharmacokinetics of Intravenous Isavuconazole in Solid-Organ Transplant Recipients.

Isavuconazole may be useful in treating and preventing fungal infections in solid-organ transplant (SOT) recipients due to its safety profile and activity against Aspergillus and some Mucorales Isavuconazole has favorable pharmacokinetics based on clinical trials in various patient populations, but data are limited in SOT recipients. We evaluated the steady-state pharmacokinetics of isavuconazole in 26 SOT recipients receiving the drug intravenously for prophylaxis. There was moderate interpatient variability in isavuconazole pharmacokinetic parameters (coefficients of variation of 51% for the area under the plasma concentration-versus-time curve [AUC] and 59% for the trough plasma concentration [Ctrough]). AUC and steady-state Ctrough were significantly lower in women, patients with a body mass index of ≥18.5 kg/m2, and those receiving hemodialysis. Trough plasma concentrations were highly correlated with AUCs (R2 = 0.94) and can serve as a suitable measure of isavuconazole exposure in patients. In conclusion, moderate interpatient variability in isavuconazole exposure, the identification of factors associated with lower exposure, the recognition that Ctrough is a surrogate marker for AUC, and the availability of a simple analytical method suggest that therapeutic drug monitoring (TDM) may be useful for guiding treatment in at least some SOT recipients. Future studies are needed to correlate isavuconazole exposure with patients' clinical outcomes and to determine the clinical role of TDM.

Changing Epidemiology of Invasive Mold Infections in Patients Receiving Azole Prophylaxis.

Breakthrough invasive mold infections (IMIs) that occur during posaconazole or voriconazole prophylaxis are rare complications for which epidemiological data are lacking. This retrospective analysis comparing 24 microbiologically documented breakthrough with 66 nonbreakthrough IMIs shows a shift towards non-Aspergillus molds with a significantly increased proportion of rare multidrug-resistant molds.

Prophylaxis with Isavuconazole or Posaconazole Protects Immunosuppressed Mice from Pulmonary Mucormycosis.

We assessed prophylactic or continuous therapy of isavuconazole, posaconazole, or voriconazole in treating pulmonary murine mucormycosis. In the prophylaxis studies, only isavuconazole treatment resulted in significantly improved survival and lowered tissue fungal burden of immunosuppressed mice infected with Rhizopus delemar. In the continuous treatment studies, isavuconazole and posaconazole, but not voriconazole, equally prolonged survival time and lowered tissue fungal burden compared to placebo-treated mice. These results support the use of isavuconazole and posaconazole in prophylaxis treatment.

New pharmacological opportunities for the treatment of invasive mould diseases.

Recently, several randomized studies have been published that will shape treatment decisions in the prevention and management of invasive mould infections. Liposomal amphotericin B is an option for empirical or targeted treatment of invasive aspergillosis or mucormycosis, but for prophylaxis therapy, the triazole class now predominates. The triazole voriconazole is currently regarded as a drug of choice for the treatment of proven or probable invasive aspergillosis, and has shown significantly higher response rates than amphotericin B deoxycholate in this setting, with fewer severe drug-related adverse events. Isavuconazole, the newest triazole agent, offers the advantages of once-daily dosing, a wider spectrum of antifungal activity than voriconazole, predictable pharmacokinetics and fewer CYP enzyme-mediated drug interactions. A recent large randomized clinical trial showed mortality to be similar under isavuconazole or voriconazole in patients with invasive mould disease, with fewer drug-related adverse events in isavuconazole-treated patients. Another study has indicated that isavuconazole is also effective in mucormycosis infections but patient numbers were small and confirmation is awaited. Experimental studies combining different drug classes with antimould activity have been promising, but the clinical database is limited. A large randomized trial of combination therapy compared voriconazole plus the echinocandin anidulafungin versus voriconazole monotherapy in patients with invasive aspergillosis. Results showed the overall response rate to be similar, but combination therapy improved survival for the subpopulation of patients in whom the diagnosis was confirmed by serum and/or bronchoalveolar lavage fluid galactomannan positivity. This active field of research is likely to continue evolving rapidly in the coming years.

Healthcare-associated outbreaks due to Mucorales and other uncommon fungi.

BACKGROUND: Healthcare-associated outbreaks of fungal infections, especially with uncommon and emerging fungi, have become more frequent in the past decade. MATERIALS AND METHOD: Here, we reviewed the history and definition of healthcare-associated outbreaks of uncommon fungal infections and discussed the principles of investigating, containing and treatment of these outbreaks. RESULTS: In case of these uncommon diseases, occurrence of two or more cases in a short period is considered as an outbreak. Contaminated medical devices and hospital environment are the major sources of these outbreaks. Care must be taken to differentiate a real infection from colonization or contamination. Defining and identifying cases, describing epidemiologic feature of cases, finding and controlling the source of the outbreak, treating patients, and managing asymptomatic exposed patients are main steps for outbreak elimination. These fungal outbreaks are not only difficult to detect but also hard to treat. Early initiation of appropriate antifungal therapy is strongly associated with improved outcomes in infected patients. Choice of antifungal drugs should be made based on spectrum, pharmacodynamic and pharmacokinetic characteristics and adverse effects of available drugs. Combination antifungal therapy and surgical intervention may be also helpful in selected cases. CONCLUSIONS: A multidisciplinary approach and close collaboration between all key partners are necessary for successful control of fungal outbreaks.

Isavuconazole therapy protects immunosuppressed mice from mucormycosis.

We studied the in vitro and in vivo efficacies of the investigational drug isavuconazole against mucormycosis due to Rhizopus delemar. Isavuconazole was effective, with MIC and minimal fungicidal concentration (MFC) values ranging between 0.125 and 1.00 µg/ml. A high dose of isavuconazole prolonged the survival time and lowered the tissue fungal burden of cyclophosphamide/cortisone acetate-treated mice infected with R. delemar and was as effective as a high-dose liposomal amphotericin B treatment. These results support the further development of this azole against mucormycosis.

Immunotherapeutic strategies against mucormycosis in haematopoietic stem cell transplantation.

Mucormycoses remain a serious complication in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT). In these patients, mortality rates of mucormycosis reach up to 90%, which is due, at least in part, to the severe and prolonged immunosuppression after transplantation. Although prolonged neutropaenia is one of the most important risk factors for mucormycosis, other cell populations, such as CD4(+) T cells may also provide critical defence mechanisms against this infection. The management of mucormycosis includes antifungal therapy, surgery and, most importantly, the control of the underlying predisposing conditions, such as the correction of an impaired immune system. Here, we review the current data of granulocytes, antifungal T cells and natural killer cells regarding their activity against mucormycetes and regarding a potential immunotherapeutic approach. It is hoped that further animal studies and clinical trials assessing immunotherapeutic strategies will ultimately improve the poor prognosis of allogeneic HSCT recipients suffering from mucormycosis.

Immunoinformatic Design of a Multivalent Peptide Vaccine Against Mucormycosis: Targeting FTR1 Protein of Major Causative Fungi.

Mucormycosis is a potentially fatal illness that arises in immunocompromised people due to diabetic ketoacidosis, neutropenia, organ transplantation, and elevated serum levels of accessible iron. The sudden spread of mucormycosis in COVID-19 patients engendered massive concern worldwide. Comorbidities including diabetes, cancer, steroid-based medications, long-term ventilation, and increased ferritin serum concentration in COVID-19 patients trigger favorable fungi growth that in turn effectuate mucormycosis. The necessity of FTR1 gene-encoded ferrous permease for host iron acquisition by fungi has been found in different studies recently. Thus, targeting the transit component could be a potential solution. Unfortunately, no appropriate antifungal vaccine has been constructed as of yet. To date, mucormycosis has been treated with antiviral therapy and surgical treatment only. Thus, in this study, the FTR1 protein has been targeted to design a convenient and novel epitope-based vaccine with the help of immunoinformatics against four different virulent fungal species. Furthermore, the vaccine was constructed using 8 CTL, 2 HTL, and 1 LBL epitopes that were found to be highly antigenic, non-allergenic, non-toxic, and fully conserved among the fungi under consideration. The vaccine has very reassuring stability due to its high pI value of 9.97, conclusive of a basic range. The vaccine was then subjected to molecular docking, molecular dynamics, and immune simulation studies to confirm the biological environment's safety, efficacy, and stability. The vaccine constructs were found to be safe in addition to being effective. Finally, we used in-silico cloning to develop an effective strategy for vaccine mass production. The designed vaccine will be a potential therapeutic not only to control mucormycosis in COVID-19 patients but also be effective in general mucormycosis events. However, further in vitro, and in vivo testing is needed to confirm the vaccine's safety and efficacy in controlling fungal infections. If successful, this vaccine could provide a low-cost and effective method of preventing the spread of mucormycosis worldwide.

In Silico Designing of a Multitope Vaccine against Rhizopus microsporus with Potential Activity against Other Mucormycosis Causing Fungi.

During the current era of the COVID-19 pandemic, the dissemination of Mucorales has been reported globally, with elevated rates of infection in India, and because of the high rate of mortality and morbidity, designing an effective vaccine against mucormycosis is a major health priority, especially for immunocompromised patients. In the current study, we studied shared Mucorales proteins, which have been reported as virulence factors, and after analysis of several virulent proteins for their antigenicity and subcellular localization, we selected spore coat (CotH) and serine protease (SP) proteins as the targets of epitope mapping. The current study proposes a vaccine constructed based on top-ranking cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B cell lymphocyte (BCL) epitopes from filtered proteins. In addition to the selected epitopes, ß-defensins adjuvant and PADRE peptide were included in the constructed vaccine to improve the stimulated immune response. Computational tools were used to estimate the physicochemical and immunological features of the proposed vaccine and validate its binding with TLR-2, where the output data of these assessments potentiate the probability of the constructed vaccine to stimulate a specific immune response against mucormycosis. Here, we demonstrate the approach of potential vaccine construction and assessment through computational tools, and to the best of our knowledge, this is the first study of a proposed vaccine against mucormycosis based on the immunoinformatics approach.

Absence of Case of Mucormycosis (March 2020-May 2021) under strict protocol driven management care in a COVID-19 specific tertiary care intensive care unit.

AIMS: With a sudden increase in cases of mucormycosis seen in Covid -19 patients, we conducted a retrospective analysis of all admitted patients in a tertiary care covid-19 hospital looking at incidence of mucormycosis. METHODS: Intensive care unit daily rounds data stored in an electronic format was retrieved by one of the consultants, looking for incidence of mucormycosis, diabetes mellitus, adherence to protocol for steroid use, glycemic control and use of monoclonal antibodies. Also, patients follow up data base of post covid Outpatients Department was searched for cases of mucormycosis. RESULTS: A total of 5248 patients were admitted between March 2020 to May 2021, of which 1027 were in ICU and 4221 in wards. Of the 1027 patients admitted in Intensive care unit, 915 received steroids and 417 had diabetes as existing co-morbidity. No case of mucormycosis was reported during the stay in the hospital and during immediate outpatient department follow up. The low dose steroids were administered as per state government protocol for treating COVID 19, a nurse driven strict glycemic control regime (blood glucose level was maintained between 140 and 180 mg/dl through the admission in ICU and was achieved consistently in 842 (82%) patients, followed along with minimal use of other immunomodulatory like monoclonal antibodies. CONCLUSION: A strict adherence to protocol of low dose steroids coupled with strict glycemic control helped in eliminating the risk and incidence of mucormycosis in a tertiary care dedicated covid-19 hospital.

Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Breakthrough zygomycosis on posaconazole prophylaxis after allogeneic stem cell transplantation.

Antifungal prophylaxis with posaconazole (POS) has been shown to decrease the mortality associated with invasive fungal infections in high-risk patients. We report on a patient, with severe graft-versus-host disease after allogeneic stem cell transplantation, who developed proven pneumonia due to Rhizopus microsporus after 40 days of POS prophylaxis (fasting serum levels: 691-904 ng/mL). Despite combination treatment with liposomal amphotericin B and POS for 39 days, the patient died from pulmonary hemorrhage. This case highlights the need for continued awareness of breakthrough zygomycosis in patients receiving POS.

Comparative immunopathogenesis in a murine model of inhalative infection with the mucormycetes Lichtheimia corymbifera and Rhizopus arrhizus.

Pathogenic mucormycetes induce diseases with considerable morbidity and mortality in immunocompromised patients. Virulence data comparing different Mucorales species and various underlying risk factors are limited. We therefore compared the pathogenesis of inhalative infection by Rhizopus (R.) arrhizus and Lichtheimia (L.) corymbifera in murine models for predominant risk factors for onset of infection. Mice with diabetes or treated with cyclophosphamide or cortisone acetate were challenged via the intranasal route with an isolate of R. arrhizus or L. corymbifera, respectively. Clinical, immunological and inflammation parameters as well as efficacy of posaconazole prophylaxis were monitored over 14 days. Whereas immunocompetent mice showed no clinical symptoms after mucormycete infection, mice treated with either cyclophosphamide (CP) or cortisone acetate (CA) were highly susceptible. Animals infected with the isolate of R. arrhizus showed prolonged survival and lower mortality, compared to those exposed to the L. corymbifera isolate. This lower virulence of R. arrhizus was risk factor-dependent, since diabetic mice died only after infection with Rhizopus, whereas all Lichtheimia-infected diabetic animals survived. Under posaconazole prophylaxis, both mucormycetes were able to establish breakthrough infections in CA- and CP-treated mice, but the course of infection was significantly delayed. Detailed analysis revealed that susceptibility of CA- and CP-treated mice could not be mimicked by exclusive lack or downmodulation of neutrophils, platelets or complement, but can be supposed to be the consequence of a broad immunosuppressive effect induced by the drugs. Both Lichtheimia corymbifera and Rhizopus arrhizus induce invasive mycoses in immunocompromised hosts after inhalative infection. Key parameters such as virulence and immunopathogenesis vary strongly according to fungal species and underlying risk group. Selected neutropenia is no sufficient risk factor for onset of inhalative mucormycosis.

Anti-inflammatory drugs selectively target sporangium development in Mucor.

It is known that acetylsalicylic acid, an anti-inflammatory and anti-mitochondrial drug, targets structure development and functions of yeasts depending on elevated levels of mitochondrial activity. Using antibody probes, we previously reported that sporangia of Mucor circinelloides also contain increased mitochondrial activity, yielding high levels of 3-hydroxyoxylipins. This was, however, not found in Mortierella alpina (subgenus Mortierella). In this study we report that acetylsalicylic acid (aspirin) also targets sporangium development of Mucor circinelloides selectively, while hyphae with lower levels of mitochondrial activity are more resistant. Similar results were obtained when the anti-inflammatory compounds benzoic acid, ibuprofen, indomethacin, and salicylic acid were tested. The anti-inflammatory drugs exerted similar effects on this dimorphic fungus as found under oxygen-limited conditions. Interestingly, sporangium development of Mortierella alpina was found not to be selectively targeted by these drugs. Mortierella alpina, which could not exhibit dimorphic growth under oxygen-limited conditions, was also more sensitive to the anti-inflammatory drugs when compared with Mucor circinelloides. These results prompt further research to assess the applicability of these antimitochondrial antifungals to protect plants and animals against Mucor infections.

Epidemiology of invasive fungal infection.

Invasive fungal infection is a growing cause of morbidity and mortality in immunosuppressed patients. Furthermore, the use of azole prophylaxis against Candida species has coincided with an increase in the incidence of invasive aspergillosis and infections by other filamentous fungi such as Mucorales. New risk factors and different timescales for onset have been identified. Knowledge of changes in the epidemiology of, and risk factors for, invasive fungal infection is particularly important when developing therapeutic strategies and effective prophylaxis to improve the prognosis of immunosuppressed patients.

Inhibition of EGFR Signaling Protects from Mucormycosis.

Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. Rhizopus species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled Rhizopus spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We performed an unbiased survey of the host transcriptional response during early stages of Rhizopus arrhizus var. delemar (R. delemar) infection in a murine model of pulmonary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis revealed activation of the host's epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon in vitro infection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with R. delemar spores. Inhibition of EGFR signaling with cetuximab or gefitinib, specific FDA-approved inhibitors of EGFR, significantly reduced the ability of R. delemar to invade and damage airway epithelial cells. Furthermore, gefitinib treatment significantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epithelial cells by R. delemar, and inhibition of EGFR signaling provides a novel approach for treating mucormycosis by repurposing an FDA-approved drug.IMPORTANCE Mucormycosis is an increasingly common, highly lethal fungal infection with very limited treatment options. Using a combination of in vivo animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs significantly increased survival following R. arrhizus var. delemar infection in mice. This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of-concept for the repurposing of FDA-approved drugs that target EGFR function.