GRP78 and Integrins Play Different Roles in Host Cell Invasion during Mucormycosis.

Mucormycosis, caused by Rhizopus species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies, or severe trauma. Inhaled Rhizopus spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here, we show that Rhizopus delemar interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cells. Expression of the two proteins is significantly enhanced by high glucose, iron, and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, R. delemar CotH7 recognizes integrin ß1 as a receptor on alveolar epithelial cells, causing the activation of epidermal growth factor receptor (EGFR) and leading to host cell invasion. Anti-integrin ß1 antibodies inhibit R. delemar invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that R. delemar interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78, thereby trapping the fungal cells within the rhinoorbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands.IMPORTANCE Mucormycosis caused by Rhizopus species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, Rhizopus causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here, we show that the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increases in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin ß1 receptor, which activates EGFR to induce fungal invasion of host cells. These results introduce a plausible explanation for disparate disease manifestations in DKA versus those in hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.

A case of invasive pulmonary aspergillosis diagnosed by transbronchial lung biopsy during treatment for diabetic ketoacidosis in a type 1 diabetic patient.

Invasive pulmonary aspergillosis (IPA) patients with non-hematological malignancy are far less than with hematological malignancy patients. We encountered a very rare case of IPA in which type 1 diabetes was the only conceivable risk factor. Further, according to the diagnostic categories of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria for IPA, the frequency of proven diagnosis is very low. Here we report a proven IPA, which rapidly developed when the patient with type 1 diabetes was being treated for diabetic ketoacidosis, which was successfully treated with the combination therapy of voriconazole (VRCZ) and micafungin (MCFG), based on early diagnosis using bronchoscopy.

Anti-CotH3 antibodies protect mice from mucormycosis by prevention of invasion and augmenting opsonophagocytosis.

Mucorales are fungal pathogens that cause mucormycosis, a lethal angioinvasive disease. Previously, we demonstrated that Rhizopus, the most common cause of mucormycosis, invades endothelial cells by binding of its CotH proteins to the host receptor GRP78. Loss of CotH3 renders the fungus noninvasive and attenuates Rhizopus virulence in mice. Here, we demonstrate that polyclonal antibodies raised against peptides of CotH3 protected diabetic ketoacidotic (DKA) and neutropenic mice from mucormycosis compared to mice treated with control preimmune serum. Passive immunization with anti-CotH3 antibodies enhanced neutrophil inlfux and triggered Fc receptor-mediated enhanced opsonophagocytosis killing of Rhizopus delemar. Monoclonal antibodies raised against the CotH3 peptide also protected immunosuppressed mice from mucormycosis caused by R. delemar and other Mucorales and acted synergistically with antifungal drugs in protecting DKA mice from R. delemar infection. These data identify anti-CotH3 antibodies as a promising adjunctive immunotherapeutic option against a deadly disease that often poses a therapeutic challenge.

Dysphagia, Hyperglycemia, and Presyncope.

There have been over 80 documented cases of swallow syncope-a rare form of reflex or neurally mediated syncope-with most cases associated with an underlying esophageal disorder. Here, we describe the first reported case of swallow syncope or presyncope caused by an infectious esophagitis. Our 65-year-old patient initially developed dysphagia, odynophagia, and presyncope with swallowing. This lead to nutrition and medication avoidance behavior, which was followed by the development of diabetic ketoacidosis. The diagnosis of swallow presyncope was confirmed with a provocative swallow study demonstrating 8 s sinus arrest, and an underlying cause of Candida esophagitis was found by upper endoscopy. Symptoms completely resolved after treatment with micafungin.

Salmonella newport causing osteomyelitis in a patient with diabetes.

Salmonella is a foodborne pathogen that commonly causes intestinal symptoms. Bacteraemia and extraintestinal infections have been documented within the literature, and are more frequently associated with immunodeficiency and general debilitation. We discuss the case of a previously well 36-year-old man who presented with a septic knee and new-onset diabetes. Imaging confirmed osteomyelitis and a Brodie's abscess, with blood and tissue cultures revealing the isolate Salmonella enterica newport. He denied any previous gastrointestinal symptoms, recent travel, change in usual dietary habit or symptoms of diabetes. So far there have only been three reported cases of S. newport causing osteomyelitis. We discuss the incidence of Salmonella infections, including extraintestinal symptoms, its relation to immunodeficiency and the disease burden of S. newport.

[Enterococcal infections: from simple to most complex]. / Infections à entérocoques: du plus simple au plus complexe...

Enterococci are microorganisms with a remar- kable ability to adapt to their environment. Two species have a significant clinical implication, Enterococcus faecalis and Enterococcus faecium. The risk factors for colonization and infection must be recognized, including prior treatment with antibiotics such as cephalosporins or quinolones. Because of their native resistance to several classes of antibiotics and the increase of acquired resistance to penicillins, the initial empiric treatment of a severe infection in a patient at risk of enterococcal infection often includes a glycopeptide. A restriction in the empirical use of cephalosporins or quinolones and a targeted antibiotic therapy following receipt of the antibiogram are essential to prevent the emergence of enterococcal strains and especially vancomycin-resistant enterococci.

Heat-killed yeast protects diabetic ketoacidotic-steroid treated mice from pulmonary mucormycosis.

Previous studies have shown that vaccination with heat-killed yeast, Saccharomyces cerevisiae (HKY), protects mice against systemic candidiasis, aspergillosis, cryptococcosis or coccidioidomycosis. Here we sought to define the potential use of HKY as a vaccine to protect mice from mucormycosis. Mice were vaccinated with different regimens of HKY prior to induction of diabetes. Diabetic ketoacidotic (DKA) mice were then treated with steroids prior to intratracheal challenge with Rhizopus oryzae. All regimens of HKY vaccine improved survival of DKA mice and reduced fungal burden in the primary target organ, lungs, as determined by qPCR. Furthermore, compared to mice vaccinated with diluent, vaccination with HKY substantially increased the mouse immune response as determined by detection of increased anti-Rhizopus antibody titers. Our results show that HKY protects steroid-treated DKA mice from pulmonary R. oryzae infection. Considering its demonstrated efficacy against other fungal infections, HKY is a promising candidate for development as a panfungal vaccine.

CotH3 mediates fungal invasion of host cells during mucormycosis.

Angioinvasion is a hallmark of mucormycosis. Previously, we identified endothelial cell glucose-regulated protein 78 (GRP78) as a receptor for Mucorales that mediates host cell invasion. Here we determined that spore coat protein homologs (CotH) of Mucorales act as fungal ligands for GRP78. CotH proteins were widely present in Mucorales and absent from noninvasive pathogens. Heterologous expression of CotH3 and CotH2 in Saccharomyces cerevisiae conferred the ability to invade host cells via binding to GRP78. Homology modeling and computational docking studies indicated structurally compatible interactions between GRP78 and both CotH3 and CotH2. A mutant of Rhizopus oryzae, the most common cause of mucormycosis, with reduced CotH expression was impaired for invading and damaging endothelial cells and CHO cells overexpressing GRP78. This strain also exhibited reduced virulence in a diabetic ketoacidotic (DKA) mouse model of mucormycosis. Treatment with anti-CotH Abs abolished the ability of R. oryzae to invade host cells and protected DKA mice from mucormycosis. The presence of CotH in Mucorales explained the specific susceptibility of DKA patients, who have increased GRP78 levels, to mucormycosis. Together, these data indicate that CotH3 and CotH2 function as invasins that interact with host cell GRP78 to mediate pathogenic host-cell interactions and identify CotH as a promising therapeutic target for mucormycosis.

[Clinical and radiological aspects of mucormycosis]. / Aspects cliniques et radiologiques des mucormycoses.

Mucormycosis is an infection caused by filamentous fungi of the Mucorales order. The predisposing factors are mostly diabetic ketoacidosis and severe immunosuppressive conditions such as prolonged neutropenia, steroid or T-cell suppressor therapy, solid organ transplantation or allogeneic hematopoietic stem cell transplantation. Mucormycosis can also occur in immunocompetent patients, especially after trauma, burns or direct inoculation of the fungi (e.g. intravenous drug abuse). The most frequently targeted primary sites of infection are sinuses with a rapid spread to the adjacent tissues including the brain, the lower respiratory tract, the digestive tract and the skin. Mucorales are able to invade the vessels causing hematogenous dissemination, vascular thrombosis and, ultimately, necrosis of the lesions. Clinical and radiological aspects are similar to those observed in other invasive filamentous fungi infections such as invasive aspergillosis, fusariosis or scedosporiosis. CT-scan or MRI are mandatory to assess the extension of the lesions. The diagnosis remains difficult and is often delayed resulting in a poor outcome.

Pathogenesis of mucormycosis.

Mucormycosis is a life-threatening infection that occurs in patients who are immunocompromised because of diabetic ketoacidosis, neutropenia, organ transplantation, and/or increased serum levels of available iron. Because of the increasing prevalence of diabetes mellitus, cancer, and organ transplantation, the number of patients at risk for this deadly infection is increasing. Despite aggressive therapy, which includes disfiguring surgical debridement and frequently adjunctive toxic antifungal therapy, the overall mortality rate is high. New strategies to prevent and treat mucormycosis are urgently needed. Understanding the pathogenesis of mucormycosis and the host response to invading hyphae ultimately will provide targets for novel therapeutic interventions. In this supplement, we review the current knowledge about the virulence traits used by the most common etiologic agent of mucormycosis, Rhizopus oryzae. Because patients with elevated serum levels of available iron are uniquely susceptible to mucormycosis and these infections are highly angioinvasive, emphasis is placed on the ability of the organism to acquire iron from the host and on its interactions with endothelial cells lining blood vessels. Several promising therapeutic strategies in preclinical stages are identified.

The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis.

Rhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first-line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron-depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidosis (DKA) mice. In addition, we disrupted FTR1 by double cross-over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti-Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus, FTR1 is a virulence factor for R. oryzae, and anti-Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis.

Subclavian artery occlusion and pseudoaneurysm caused by lung apex mucormycosis: successful treatment with transcatheter embolization.

Subclavian artery pseudoaneurysm and occlusion in young patients are usually post-traumatic. We report the case of a 33-year-old diabetic woman with subclavian artery occlusion and pseudoaneurysm formation caused by pulmonary mucormycosis infection. The patient presented with diabetic ketoacidosis, Horner's syndrome, and absent left arm pulses. A cystic lesion of the left lung apex was found by imaging, was surgically resected, and was histologically diagnosed as mucormycosis infection. Magnetic resonance angiography depicted a left subclavian artery pseudoaneurysm and occlusion adjacent to the mucormycosis lesion. To protect against thromboembolic complications and rupture, the pseudoaneurysm was embolized with coils. The patient is clinically well 1 year after the intervention with no perfusion of the pseudoaneurysm.

Deferiprone iron chelation as a novel therapy for experimental mucormycosis.

OBJECTIVES: Patients treated with the iron chelator deferoxamine are known to be more susceptible to mucormycosis. However, while deferoxamine is an iron chelator from the perspective of the human host, deferoxamine actually serves as a siderophore, delivering free iron to Rhizopus oryzae, the major cause of mucormycosis. Other iron chelators, including deferiprone, which do not deliver iron to R. oryzae have been described. We therefore sought to determine whether iron-chelation therapy with deferiprone would effectively treat mucormycosis. METHODS: In vitro MIC and minimum fungicidal concentration (MFC) of the iron chelator, deferiprone, for R. oryzae were determined by microdilution assay. In addition, we compared the efficacy of deferiprone with that of liposomal amphotericin B (LAmB) in treating mucormycosis in diabetic ketoacidotic mice. RESULTS: Deferiprone demonstrated static activity against R. oryzae at 24 h, but showed cidality at 48 h of incubation. Deferiprone was as effective as LAmB at improving survival and decreasing brain fungal burden, and both drugs were more effective than placebo in non-iron-overloaded animals. Administration of free iron with deferiprone reversed protection, confirming that the mechanism of protection was iron chelation. CONCLUSIONS: Iron chelation is a promising, novel therapeutic strategy for refractory mucormycosis infections. Further studies are warranted to evaluate combination antifungal/iron chelation therapy and to evaluate the efficacy of other iron-chelating agents.

Rate and prediction of infection in children with diabetic ketoacidosis.

The purpose of this retrospective cohort study was to determine the rate and prediction of infection in children, < or = 21 years, with diabetic ketoacidosis (DKA). Over a 6-year period, 247 admissions were identified. There were 171 (69%) with no infection, 44 (17.8%) with presumed viral infection, and 32 (12.9%) with bacterial infection. The mean WBC for all patients was 17,519 ( +/- 9,582). 118 (50%) had leukocytosis as defined by a WBC > or = 15,000/mm3. WBC, differential, leukocytosis, as well as sex, temperature and new onset diabetes, were not significant predictors (P > .05) of bacterial infection. Bacterial infections were more common in children < or = 3 years of age (P = .03). There was a significant correlation of WBC with both pH (r = -0.59, P < .001) and bicarbonate (r = -0.43, P < .001). In conclusion, most children in DKA have no evidence of infection. Leukocytosis is common but most likely reflects the severity of DKA rather than the presence of infection.

Pyogenic liver abscesses due to Klebsiella pneumoniae in a diabetic patient.

Pyogenic liver abscess due to Klebsiella pneumoniae is a rare clinical entity. It has emerged as an important infection complication in diabetics and its incidence in diabetics without intraabdominal or biliary tract infections is increasing. We present herein a case of multiple pyogenic liver abscesses due to K. pneumoniae in a diabetic patient and discuss clinical course, treatment and possible reasons for association between K. pneumoniae liver abscess and diabetes.

Infection as a trigger of diabetic ketoacidosis in intensive care-unit patients.

We determined the prevalence and indicators of infection in intensive care unit (ICU) patients with diabetic ketoacidosis (DKA) by performing a retrospective analysis of 123 episodes of DKA (in 113 patients) managed in a medical ICU between 1990 and 1997. In univariate analysis, features associated with infection were female sex, neurological symptoms at admission, fever during the week before admission, a need for colloids, a high blood lactate level at admission, and lack of complete clearance of ketonuria within 12 h. Multivariate analysis identified 3 independent predictors of infection: female sex (odds ratio [OR], 2.31; confidence interval [CI], 1.05-5.35), neurological symptoms at admission (OR, 2.83; CI, 1.18-6.8), and lack of complete clearance of ketonuria within 12 h (OR, 3.73; CI, 1.58-9.09). Infection is the leading trigger of DKA in ICU patients. Neurological symptoms at admission and lack of complete clearance of ketonuria within 12 h are useful warning signals of infection.

[Investigation of the form of onset in 139 cases of insulin-dependent diabetes mellitus].

A retrospective study of 139 cases of IDDM patients (male 54 cases, female 85 cases) were investigated. Average age 31.74 +/- 12.20 yr. One third of all patients had virus infection before the onset of IDDM and the infection in upper respiratory system and intestine occupied most of the cases (85%). In two cases, the onset of the disease was induced after BCG vaccination. There is no relationship between virus infection and the occurrence of DKA as primary form of onset. The average age in the group of positive virus infection is significantly younger than in the group of negative virus infection (P < 0.02). About one fourth of all cases had positive family history of diabetes which is much higher than the similar reports in Western countries.