The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae.

Background: In response to the replace mammal research models with insects in preliminary immunological studies, interest has grown in invertebrate defense systems. The immunological response is regulated by cytokines; however, while their role in mammals is well understood, little is known of their function in insects. A suitable target for studies into insect immunology is Galleria mellonella (Lepidoptera), the wax moth: a common host for human fungal and bacterial pathogens. G. mellonella is also a perfect subject for studies into the presence of cytokine-like proteins. Specific objectives: The main goal of present research was detection in insect immunocompetent cells the 18 mammalian cytokines (IL-1α, IL-1ß, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-ß, GM-CSF, M-CSF, G-CSF), which play important role in immunological response and indication how their level change after fungal infection. Methodology: The changes of cytokine-like proteins level were detected in hemocytes taken from G. mellonella larvae infected with entomopathogenic fungus, C. coronatus. The presence of cytokine-proteins was confirmed with using fluorescence microscopy (in cultured hemocytes) and flow cytometry (in freshly collected hemolymph). The ELISA test was used to detect changes in concentration of examined cytokine-like proteins. Results: Our findings indicated the presence of eighteen cytokine-like molecules in G. mellonella hemocytes during infection with C. coronatus. The hemocytes taken from infected larvae demonstrated higher fluorescence intensity for six cytokine-like proteins (GM-CSF, M-CSF, IL-3, IL-15, IL-1ß and IL-19) compared to untreated controls. ELISA test indicated significantly higher IL-3 and IL-15. M-CSF, IL-1α and IL-19 concentration in the hemolymph after fungal infection, and significantly lower TNF-ß and G-CSF. Conclusions: Our findings confirm that the selected cytokine-like molecules are present in insect hemocytes and that their concentrations change after fungal infection, which might suggest that they play a role in the anti-fungal immunological response.

Clinical characteristics of 5 adult patients with rhinofacial entomophthoromycosis in Thailand.

Five patients [mean age: 36 years (range: 22-65)] were diagnosed with rhinofacial entomophthoromycosis at our center during the study period. All patients presented with painless cheek and nasal dorsum swelling with nasal obstruction. All pathology reports confirmed rhinofacial entomophthoromycosis, which is characterized by the Splendore-Hoeppli phenomenon. Conidiobolus coronatus was identified from fungal culture in all patients. All patients were successfully treated with various antifungals.

The Entomopathogenic Fungus Conidiobolus coronatus Has Similar Effects on the Cuticular Free Fatty Acid Profile of Sensitive and Resistant Insects.

The mechanisms underlying the recognition of a susceptible host by a fungus and the role of cuticular compounds (CCs) in this process remain unclear; however, accumulated data suggest that this is influenced to a great degree by cuticular lipids. Two insect species differing in their sensitivity to fungal infection, viz. the highly sensitive Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) and the resistant Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae), exhibited significant qualitative and quantitative changes in cuticular free fatty acid (FFA) profiles after exposure to Conidiobolus coronatus (Constantin) Batko (Entomopthorales). Despite being systematically distant, leading different lifestyles in different habitats, both insect species demonstrated similar changes in the same FFAs following exposure to the fungus (C12:0, C13:0, C14:0, C15:0, C16:1, C16:0, C18:1, C18:0), suggesting that these are involved in a contact-induced defense response. As it was not possible to distinguish the share of FFAs present in the conidia that were attached to the cuticle from the FFAs of the cuticle itself in the total number of extracted FFAs, further research is necessary.

A rare case of condiobolomycosis due to Conidiobolus coronatus presenting with dysphagia.

Conidiobolomycosis is a neglected tropical fungal infection involving the head and neck region. Here we report the first case of atypical conidiobolomycosis presenting with dysphagia and significant weight loss from Odisha, India. It was diagnosed by histopathology and fungal culture and was suscessfully treated with saturated solution of potassium iodide.

Infection of Galleria mellonella (Lepidoptera) Larvae With the Entomopathogenic Fungus Conidiobolus coronatus (Entomophthorales) Induces Apoptosis of Hemocytes and Affects the Concentration of Eicosanoids in the Hemolymph.

Apoptosis and autophagy, the mechanisms of programmed cell death, play critical roles in physiological and pathological processes in both vertebrates and invertebrates. Apoptosis is also known to play an important role in the immune response, particularly in the context of entomopathogenic infection. Of the factors influencing the apoptotic process during infection, two of the lesser known groups are caspases and eicosanoids. The aim of this study was to determine whether infection by the entomopathogenic soil fungus Conidiobolus coronatus is associated with apoptosis and changes in caspase activity in the hemocytes of Galleria mellonella larvae, and to confirm whether fungal infection may affect eicosanoid levels in the host. Larvae were exposed for 24 h to fully grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24 group) or 24 h later (F48 group). Apoptosis/necrosis tests were performed in hemocytes using fluorescence microscopy and flow cytometry, while ELISA tests were used to measure eicosanoid levels. Apoptosis and necrosis occurred to the same degree in F24, but necrosis predominated in F48. Fungal infection resulted in caspase activation, increased PGE1, PGE2, PGA1, PGF2α, and 8-iso-PGF2α levels and decreased TXB2 levels, but had no effect on TXA2 or 11-dehydro-TXB2 concentrations. In addition, infected larvae demonstrated significantly increased PLA2 activity, known to be involved in eicosanoid biosynthesis. Our findings indicate that fungal infection simultaneously induces apoptosis in insects and stimulates general caspase activity, and this may be correlated with changes in the concentrations of eicosanoids.

Entomophthoramycosis: An unusual cause of facial disfigurement.

Entomophthoramycosis is a rare fungal infection of nose, paranasal sinuses and subcutaneous tissues found in tropical and subtropical region. From India very few cases have been reported. Here we report a case of Entomophthoramycosis due to Conidiobolus coronatus from the eastern India who presented with slowly growing rhinofacial swelling and right sided nasal obstruction due to intranasal mass. The case was diagnosed by typical histopathological findings of broad aseptate hyphae with surrounding eosinophilic granular material (Splendore Hoeppli phenomenon) on microscopy of nasal biopsy material and confirmed by PCR assay of DNA and sequencing from biopsy tissue. Treatment with saturated solution of potassium iodide and itraconazole was successful and clinical cure was attained in 8 months.

Nasofacial rhinoentomophthoramycosis: A report of two cases from Central India.

Rhinoentomophthoromycosis due to Conidiobolus coronatus is a rare, chronic, granulomatous disease, occurring mainly in tropical countries including India. We report two cases of rhinoentomophthoromycosis in an 18-year-old female and a farmer of 35 years residents of Chhattisgarh shifted from Madhya Pradesh and Orrisa. It was diagnosed by microscopy and isolation C. coronatus in culture. The patient presented with a swollen nose with obstruction that had progressed slowly over 1 year. His nasal swelling was bilateral, diffuse, mildly tender, erythematous, nonpitting, with mucosal crusting and hypertrophy of inferior turbinates but no regional lymphadenopathy. Culture of tissue from the nasal biopsy on sabouraud dextrose agar yielded multiple colonies of a mold with satellite smaller colonies at periphery. The isolate demonstrated the macroscopic and microscopic morphologic characteristics of C. coronatus. The patients were earlier treated with itraconazole or its combination with potassium iodide and the patients were treated successfully with amphotericin B.

Chronic invasive rhinosinusitis by Conidiobolus coronatus, an emerging microorganism.

Fungi from the Conidiobolus genus have been implicated in the development of chronic invasive fungal rhinosinusitis, mainly in tropical countries. The mycosis associated to these fungi may cause irreversible facial deformities and is potentially fatal. The authors present the first case of a chronic invasive fungal rhinosinusitis due to Conidiobolus coronatus diagnosed in a 66-year-old Caucasian male patient, living in Portugal without any travels abroad and complaining of progressive refractory nasal obstruction, facial pain and anosmia. Upon the culture of samples collected during sinus endoscopic surgery, colonies that presented a macroscopic aspect remembering wax were detected. The microscopic evaluation allowed the observation of simple conidiophores forming spherical conidia, and of conidiophores that presented hair-like appendages. Together, these characteristics allowed the identification of the fungi as a Conidiobolus, which was confirmed upon the DNA sequencing. The authors emphasised the role of this fungi as an emergent microorganism as well as the difficulties associated to the diagnosis and treatment.

Blooming of Unusual Cytochrome P450s by Tandem Duplication in the Pathogenic Fungus Conidiobolus coronatus.

While the Zygomycete fungus Conidiobolus coronatus primarily infects insects, it can be pathogenic to mammals as well, including humans. High variability in the treatment of this fungal infection with currently available drugs, including azole drugs is a very common phenomenon. Azoles bind to the cytochrome P450 monooxygenases (P450s/CYP) including CYP51, a sterol 14-α-demethylase, inhibiting the synthesis of cell membrane ergosterol and thus leading to the elimination of infecting fungi. Despite P450's role as a drug target, to date, no information on C. coronatus P450s has been reported. Genome-wide data mining has revealed the presence of 142 P450s grouped into 12 families and 21 subfamilies in C. coronatus. Except for CYP51, the remaining 11 P450 families are new (CYP5854-CYP5864). Despite having a large number of P450s among entomopathogenic fungi, C. coronatus has the lowest number of P450 families, which suggests blooming P450s. Further analysis has revealed that 79% of the same family P450s is tandemly positioned, suggesting that P450 tandem duplication led to the blooming of P450s. The results of this study; i.e., unravelling the C. coronatus P450 content, will certainly help in designing experiments to understand P450s' role in C. coronatus physiology, including a highly variable response to azole drugs with respect to P450s.

A first-line antiretroviral therapy-resistant HIV patient with rhinoentomophthoromycosis.

The Conidiobolus coronatus-related rhinoentomophthoromycosis in immunocompetent and immunocompromised (HIV negative) individuals has been treated successfully with antifungal drugs. However, C. coronatus infections in first-line antiretroviral therapy (ART)-resistant (HIV infected) individuals particularly with rhinoentomophthoromycosis have not been reported previously. Here, we describe a case of itraconazole non-responding rhinoentomophthoromycosis in an HIV-infected patient with first-line antiretroviral (ART) drug resistance which was successfully managed through systematic diagnostic and therapeutic approaches in dermatologic setting. A 32-year-old HIV-1-infected man presented with painless swelling, nasal redness and respiratory difficulty. The patient was receiving first-line ART and had a history of traumatic injury before the onset of nasopharyngeal manifestations. The patient's previous history included oral candidiasis and pulmonary tuberculosis.

Look before you leap: A case series of conidiobolomycosis from the Indian subcontinent.

Conidiobolomycosis is an extremely rare subcutaneous rhinofacial indolent infection caused by Conidiobolus coronatus and Conidiobolus incongruus. It is reported mainly from the tropical and subtropical parts of the world, mostly in form of isolated case reports or small case series. Two immunocompetent male patients presented to our center with indolent nasal swelling and features of nasal obstruction. They were treated outside with antibiotics and/or steroids and were referred to us with nonresolving symptoms. Both of them were diagnosed based on the characteristic histopathological findings and were successfully treated with saturated solution of potassium iodide. Conidiobolomycosis is an important differential in patients presenting with centrofacial swelling. Early suspicion and timely referral by the primary care physicians will help in early diagnosis and treatment of this disease.

Pathogenicity of Conidiobolus coronatus and Fusarium solani in mouse models.

To study the pathogenicity of Conidiobolus coronatus (C. coronatus) and Fusarium solani (F. solani) in animal models. Immunocompromised mice were treated with cyclophosphamide and prednisolone via intraperitoneal injection before and after inoculation. According to pathogenic characteristics of different fungi, C. coronatus was used to infect mice via intravenous inoculation, intraperitoneal inoculation, gastrointestinal infusion and intradermal inoculation methods. And F. solani was used to infect mice by inoculation via the abraded or normal skin. In the group of immunocompromised mice, C. coronatus was isolated from the lung tissues of one mouse on day 7 and another on day 10 respectively. The corresponding histopathology revealed infiltration of local inflammatory cells in the lung tissue. Pathogenic lesions were observed in all normal and immunocompromised mice infected with F. solani via abraded skin. The lesions in the immunocompromised mice were more severe and persisted longer than those in the normal mice. Moreover, hyphae were mostly observed in the histopathological examination and fungal culture from the immunocompromised mouse. The pathogenicity of C. coronatus was relatively weak as it did not induce local infections and did not disseminate the disease in immunocompetent and immunocompromised mice. Therefore, F. solani is a type of opportunistic pathogenic fungus, and abraded skin is one of the causative routes of infection.

Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus.

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus-cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.

Effect of Conidiobolus coronatus on the Cuticular and Internal Lipid Composition of Tettigonia viridissima Males.

Conidiobolus coronatus is an entomopathogenic fungus which has a potential as a biological control agent of insects. The cuticular and internal lipid composition of infected and noninfected Tettigonia viridissima males were analyzed by GC/MS. A total of 49 compounds were identified in the infected and noninfected males, including fatty acids, fatty acid methyl esters (FAMEs), n-alkanes, alcohols, sterols, and other organic compounds. The most abundant components of the cuticular and internal lipids of the insects were fatty acids. After exposure to C. coronatus, the cuticular lipids of the T. viridissima males contained 17 free fatty acids from C(8) to C(22), while the cuticular lipids of the noninfected insects contained only 15 fatty acids from C(12) to C(24). The cuticular and internal lipids of both the infected and the noninfected males also contained five FAMEs from C(15) to C(19), seven n-alkanes from C(25) to C(34), five alcohols from C(16) to C(25), five sterols, and the following six other organic compounds: azelaic acid, phenylacetic acid, glutaric acid, benzoic acid, sebacic acid, and glycerol. The compounds which were present only in the cuticular lipids of the infected males could be due to fungal infection.

[Rhinofacial conidiobolomycosis associated with cervical, thoracic and brachial localizations: one clinical case in Nigeria]. / Conidiobolomycose rhinofaciale avec localisations cervicales, thoraciques et brachiales: à propos d'un cas nigérian.

We report here the clinical case of a Nigerian adult patient who received medical care during October 2010, at the Center for Diagnosis and Treatment of Buruli ulcer in Pobè (Benin). He presented a massive facial tumor associated with several subcutaneous (cervical, thoracic and upper limbs) nodules, evolving since several years. Tissue samples collected at Pobè medical center were addressed to the mycology and histology laboratories of Angers University Hospital (France), according to the medical exchange agreement between the two institutions about the diagnosis and treatment of Buruli ulcer disease. Histological examination showed a Splendore-Hoeppli phenomenon, consisting of a granulomatous reaction made of eosinophilic polynuclear cells surrounding rare, large and irregular, non-septate hyphae. A filamentous fungus was isolated by cultivation of the clinical samples, which was identified as Conidiobolus coronatus. The patient was treated orally with daily doses of ketoconazole (400 mg per day). After 4 months of treatment, a marked regression of the facial lesion was obtained. A first constructive facial surgery was achieved, but the patient did not attend the second step. This case report allows us to remind the mycological diagnosis of this exotic mycosis, but also to emphasize the main difficulties encountered in medical management in the developing countries.

Rhinocerebral and rhinopharyngeal conidiobolomycosis in sheep / Conidiobolomicose rinocerebral e rinofaríngea em ovinos

The present study aimed to report the occurrence as well as the clinical, laboratorial, pathological and mycological findings of three outbreaks of rhinocerebral and rhinopharyngeal conidiobolomycosis in sheep from Rio Grande do Norte state, Brazil. A total of six sheep with clinical signs of conidiobolomycosis were evaluated, and information on history was obtained from ranchers. Clinical signs included depression, progressive emaciation, sero-sanguinolent nasal discharge, tachycardia, dyspnea, miosis and unilateral exophthalmia with increased ocular globe volume, corneal ulceration and nervous system signs. The main hematological alteration was neutrophilia. Serum biochemical evaluation revealed increased values for AST, GGT, creatinine, urea, and glucose, and decreased total proteins and albumin levels. Cerebrospinal fluid analysis showed the presence of fibrin reticules and pleocytosis. Upon necropsy, longitudinal sections of the head revealed the presence of a nodular mass with a friable consistency and a white-yellowish coloration. Microscopic findings included meningitis, cortex necrosis and encephalitis with the presence of eosinophilic Splendore-Hoeppli substance. Histopathology of the lungs revealed a Splendore-Hoeppli-like material and hyperplasia of alveolar and bronchiolar epithelium. Renal lesions were suggestive of amyloidosis.

O presente estudo objetivou relatar a ocorrência e os achados clínicos, laboratoriais, patológicos e micológicos de três surtos de conidiobolomicose rinocerebral e rinofaríngea em ovinos do estado do Rio Grande do Norte, Brasil. Um total de seis ovinos com sinais clínicos de conidiobolomicose foi avaliado, e informações sobre o histórico foram obtidas dos proprietários. Os sinais clínicos incluíram depressão, emaciação progressiva, descarga nasal serossanguinolenta, taquicardia, dispneia, miosite e exoftalmia unilateral com aumento do volume do globo ocular, ulceração da córnea e sinais nervosos. A principal alteração hematológica foi neutrofilia. A avaliação bioquímica sérica revelou aumento nos valores de AST, GGT, creatinina, ureia e glicose, e redução nos níveis de proteínas totais e albumina. A análise do fluido cerebrospinal revelou presença de retículos de fibrina e pleocitose. À necropsia, secções longitudinais da cabeça revelaram presença de uma massa nodular com consistência friável e coloração branco-amarelada. Os achados microscópicos incluíram meningite, necrose do córtex e encefalite com presença da substância eosinofílica de Splendore-Hoeppli. A histopatologia dos pulmões revelou uma substância similar à de Splendore-Hoeppli e hiperplasia do epitélio alveolar e bronquiolar. As lesões renais foram sugestivas de amiloidose.

Zigomicose nasofacial no Estado do Pará: registro de dois casos / Nasofacial zygomycosis in the state of Pará: the registration of 2 cases

São descritos dois casos de zigomicose nasofacial, procedentes de Belém, PA. O achado indica não ser tão rara, como se poderia supor, pela falta de registros, a ocorrência dessa micose na Região Norte do Brasil. Em ambos os pacientes, o tratamento pelo cetoconazol determinou involução rápida das manifestações clínicas, principalmente a obstrução nasal. Com o presente relato, sobe para cinco o número de casos da infecção referidos até agora do Estado do Pará.

Two new cases of nasofacial zygomycosis from the state of Pará, Brazil, are reported. Both cases were treated with ketoconazole and the response to the drug was considered to be good; the patients improved rapidly and the nasal obstruction was the first manifestation to disappear. As five cases have been described in recent years from the state of Pará, this form of zygomycosis can no longer be considered as a rare disease in northern Brazil.