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.

Heat shock proteins (HSP 90, 70, 60, and 27) in Galleria mellonella (Lepidoptera) hemolymph are affected by infection with Conidiobolus coronatus (Entomophthorales).

Invertebrates are becoming more popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. The humoral immune response in insects acts by melanization, clotting and the production of reactive oxygen species and antimicrobial peptides, while the cellular immunity system is based on nodulation, encapsulation and phagocytosis. An increasingly popular insect model in biological research is Galleria mellonella, whose larvae are sensitive to infection by the entomopathogenic fungus Conidiobolus coronatus, which can also be dangerous to humans. One group of factors that modulate the response of the immune system during infection in mammals are heat shock proteins (HSPs). The aim of this study was to investigate whether infection by C. coronatus in G. mellonella hemolymph is accompanied by an increase of HSP90, HSP70, HSP60 and HSP27. Larvae (five-day-old last instar) were exposed for 24 hours to fully-grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24) or 24 hours later (F48). The concentration of the HSPs in hemolymph was determined using ELISA. Immunolocalization in hemocytes was performed using fluorescence microscopy and flow cytometry. HSP90, HSP70, HSP60 and HSP27 were found to be present in the G. mellonella hemocytes. HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 being found in trace amounts; HSP60 and HSP27 were elevated in F24 and F48, and HSP90 was elevated in F48. The fungal infection had no effect on HSP70 levels. These findings shed light on the mechanisms underlying the interaction between the innate insect immune response and entomopathogen infection. The results of this innovative study may have a considerable impact on research concerning innate immunology and insect physiology.

Human Pathogenic Entomophthorales.

The pathogenic entomophthoralean fungi cause infection in insects and mammalian hosts. Basidiobolus and Conidiobolus species can be found in soil and insect, reptile, and amphibian droppings in tropical and subtropical areas. The life cycles of these fungi occur in these environments where infecting sticky conidia are developed. The infection is acquired by insect bite or contact with contaminated environments through open skin. Conidiobolus coronatus typically causes chronic rhinofacial disease in immunocompetent hosts, whereas some Conidiobolus species can be found in immunocompromised patients. Basidiobolus ranarum infection is restricted to subcutaneous tissues but may be involved in intestinal and disseminated infections. Its early diagnosis remains challenging due to clinical similarities to other intestinal diseases. Infected tissues characteristically display eosinophilic granulomas with the Splendore-Höeppli phenomenon. However, in immunocompromised patients, the above-mentioned inflammatory reaction is absent. Laboratory diagnosis includes wet mount, culture serological assays, and molecular methodologies. The management of entomophthoralean fungi relies on traditional antifungal therapies, such as potassium iodide (KI), amphotericin B, itraconazole, and ketoconazole, and surgery. These species are intrinsically resistant to some antifungals, prompting physicians to experiment with combinations of therapies. Research is needed to investigate the immunology of entomophthoralean fungi in infected hosts. The absence of an animal model and lack of funding severely limit research on these fungi.

Zygomycotic infective endocarditis in pregnancy.

Under the circumstances of cardiovascular adaptations and immunomodulation, an uncommon but disastrous complication of infective endocarditis (IE) can occur in pregnancy. Almost all the cases reported earlier were caused by bacteria. We report a fatal case of zygomycotic valvular and mural endocarditis in a young non-diabetic primigravida with a positive hepatitis B serology.

Visceral basidiobolomycosis: An overlooked infection in immunocompetent children.

Visceral basidiobolomycosis is an unusual fungal infection of viscera caused by saprophyte Basidiobolus ranarum. It is very rare in healthy children and poses a diagnostic challenge due to the non-specific clinical presentation and the absence of predisposing factors. We report a case of gastrointestinal basidiobolomycosis in a 4-year-old healthy girl who presented with a short history of abdominal pain, bleeding per rectum, fever, and weight loss. The diagnosis was based on high eosinophilic count, classical histopathology findings of fungal hyphae (the Splendore-Hoeppli phenomenon), and positive fungal culture from a tissue biopsy. Fungal infection was successfully eradicated with a combined approach of surgical resection of the infected tissue and a well-monitored course of antifungal therapy. The atypical clinical presentation, diagnostic techniques, and the role of surgery in the management of a rare and lethal fungal disease in an immunocompetent child are discussed.

Fascitis necrotizante por Saksenaea vasiformis en una paciente inmunocompetente tras un accidente de tráfico / Necrotising fasciitis caused by Saksenaea vasiformis in an immunocompetent patient after a car accident

Antecedentes. La mucormicosis (zigomicosis) cutánea con extensión subcutánea y diseminación en pacientes inmunocompetentes es una patología infrecuente causada por especies de los géneros Apophysomyces, Rhizopus y Saksenaea, entre otros. Caso clínico. Se describe un caso de fascitis necrotizante por Saksenaea vasiformis en una mujer inmunocompetente, que sufrió politraumatismo y herida en brazo derecho a consecuencia de un accidente de tráfico. Tras la reducción quirúrgica de las fracturas, la lesión cutánea fue empeorando y derivó en necrosis y fascitis necrotizante grave con cultivos inicialmente negativos. A pesar de la amplia resección quirúrgica y el tratamiento antifúngico agresivo, el desenlace fue la muerte de la paciente. El estudio histopatológico puso en evidencia una infección por un hongo del orden Mucorales, confirmada mediante aislamiento primario en agar Sabouraud y posterior identificación de la especie mediante cultivo en agar Czapek-Dox y secuenciación de la región ITS del ADN ribosomal. Conclusiones. El presente caso confirma la presencia de este hongo en nuestro país, además de la utilidad del estudio histopatológico para el diagnóstico de la mucormicosis, y de los medios de cultivo especiales y las técnicas moleculares para la identificación de la especie(AU)

Background. Cutaneous mucormycosis (zygomycosis), with subcutaneous spreading and dissemination, in immunocompetent patients is an uncommon disease caused by species belonging to the fungal genera Apophysomyces, Rhizopus and Saksenaea, among others. Case report. A case of necrotising fasciitis by Saksenaea vasiformis in an immunocompetent woman is described. The infection was acquired through a car accident resulting in multiple injuries affecting mainly her right arm. After the surgical reduction of fractures, skin lesions worsened and led to necrosis. The patient quickly developed a severe necrotising fasciitis with negative cultures at first. Despite the extensive surgical debridement and the aggressive antifungal treatment, the patient died. The histopathological study showed a fungal infection due to a fungus belonging to the Mucorales order, which was confirmed by culturing the clinical sample on Sabouraud agar, and identifying the species by cultures on Czapek-Dox agar, and sequencing of the ITS region of the ribosomal DNA. Conclusions. This case confirm the presence of this fungus in Spain, the value of histopathology for the mucormycosis diagnosis, as well as the need to perform special cultures to facilitate their isolation and identification to the species level by the combined use of Czapek-Dox agar and sequencing of the ITS region(AU)

Successful treatment of cutaneous zygomycosis with extensive surgical debridement and oral posaconazole in an immunocompetent patient.

We describe a case of successful treatment of cutaneous zygomycosis using a combination of extensive surgical debridement and the oral antifungal agent, posaconazole.

Host defenses against zygomycetes.

Mucormycosis is a devastating disease and can occur in patients with a variety of risk factors, the most important of which are immunosuppression, anatomic barrier breakdown, iron overload, and hyperglycemia/acidosis. Similarly to what occurs with Aspergillus, the host stimulates an innate immune response against the challenging sporangiospores and invading hyphae of Zygomycetes. This article discusses the host defense to different Zygomycetes, its augmentation, and its subsequent impact on the outcome of mucormycosis.

Pneumonia and lung infections due to emerging and unusual fungal pathogens.

Invasive mold infections affecting the lungs are increasing in incidence and diversity. Severely immunocompromised patients are particularly vulnerable to infection from unusual, normally nonpathogenic fungi that are found naturally in the environment. Certain fungi such as Scedosporium and the dematiaceous fungi also cause lung disease in hosts without overt immune compromise. The impacts of these emerging pathogens range from airway colonization to locally invasive lung, and disseminated, disease. Diagnosis requires isolation and identification of the etiologic agent by either or both phenotypic and molecular biology methods. Evidence of tissue invasion on histopathology is often required to distinguish infection from colonization. Diagnostic imaging techniques are nonspecific, and there are no reliable serological biomarkers of infection. Many rare molds and yeasts demonstrate reduced in vitro susceptibility to antifungal agents. Although amphotericin B formulations remain clinically useful for many of these infections, voriconazole and posaconazole are more effective for some of these difficult-to-treat pathogens. Surgical resection of diseased tissue and support of the host immune system are often required to optimize outcomes.

Documento de consenso de la Sociedad de Infectología Pediátrica sobre el tratamiento de la infección fúngica basado en la respuesta inmunológica / Spanish Paediatric Infectious Diseases Society Consensus Document on the Treatment of Fungal Infections Based on the Immune Response

La infección fúngica invasiva es una infección de tipo oportunista que afecta principalmente al paciente inmunodeprimido y crítico y que, a pesar de los avances en el diagnóstico y tratamiento, sigue comportando una elevada morbimortalidad. Esto ha determinado la investigación de tratamientos coadyuvantes a la terapia antifúngica estándar. Entre ellos, destaca el tratamiento basado en la respuesta inmunológica, que comprende el tratamiento inmunomodulador (transfusión de células T y de células dendríticas, factores estimuladores de colonias, interferón gamma, interleucina 12, vacunas antifúngicas, factores de transferencia y ciertos fármacos como la cloroquina) y el tratamiento inmunoterápico que incluye la transfusión de granulocitos, los anticuerpos monoclonales y la inmunoglobulina endovenosa. El presente documento recoge una revisión y actualización de los datos disponibles sobre esta modalidad terapéutica y aporta los conocimientos básicos de la respuesta inmune frente a la infección fúngica para poder comprender mejor el papel de dicha estrategia terapéutica en la respuesta al tratamiento antifúngico convencional, así como sus potenciales indicaciones en el paciente pediátrico. Existen pocos datos sobre evidencia científica y grado de recomendación para su uso (AU)

Despite the emergence of new diagnostic and therapeutic methods, invasive fungal infections are still a major cause of morbidity and mortality in immunocompromised and critical patients. Therefore, adjuvant treatments to the standard antifungal therapy are being investigated, with immunity-based therapy being one of the most important. Both immunomodulatory (dendritic and T cells transfusions, colony stimulating factors, interferón-gamma, interleukin 12, fungal vaccines, transfer factors and certain drugs such as chloroquine) and immunotherapeutic modalities (granulocyte transfusions, monoclonal antibodies and intravenous immunoglobulin) have been described. This document aims to summarise currently available data on immunity-based therapy of fungal infections and to provide basic knowledge on the immune response to fungal infections. This helps to understand how, in selected cases, immunity-based therapy may improve the response to standard antifungal treatment. The potential indications of immunity-based therapy in the paediatric patient are reviewed, although there is still a lack of scientific evidence for its use in children (AU)

Zigomicosis cutánea primaria en paciente pediátrica inmunocomprometida / Primary zygomycosis in a pediatric inmunocompromised patient

La zigomicosis es una infección infrecuente causada por hongos oportunistas que pertenecen al orden Mucorales, cuya característica es la invasión vascular por hifas, lo que determina trombosis, infarto y necrosis tisular. En General afecta a pacientes con efermedades subyacentes, causando infecciones invasoras severas y a menudo fulminantes. Algunas condiciones de riesgo son: leucemias y linfomas con neutropenia prolongada, cetoacidosis diabética, malnutrición severa, ruptura de la integridad de la barrera cutánea y terapia inmunosupresora. Se presenta un caso de zigomicosis cutánea primaria en el antebrazo, en una paciente pedíatrica con leucemia linfobástica aguda de tipo B, con grave neutropenia y tratamiento con corticoides, y describimos los aspectos histológicos y clínicos de la lesión necrótica en la zona de punción venosa.

Cutaneous zygomycosis: a possible postoperative complication in immunocompetent individuals.

Fungi in the class of zygomycetes usually produce serious infections in diabetics and immunocompromised hosts. Cutaneous zygomycosis is a less common form, with an unpredictable extent of anatomical involvement and clinical course. Here, we report two cases of primary cutaneous zygomycosis as postoperative complications in otherwise healthy females. Zygomycosis was suspected and specimens from the surgical debridement were examined by microbiological and histopathological studies for confirming the clinical diagnosis. Rapid diagnosis, liposomal amphotericin B, and proper debridement of affected tissue are necessary to avoid a fatal outcome.

The role of neutrophils in the development and outcome of zygomycosis in haematological patients.

Zygomycosis constitutes the third leading cause of invasive fungal infections following aspergillosis and candidosis. Patients with haematologic malignancies or haematopoietic stem cell transplantation are particularly susceptible to zygomycosis. Neutropenia represents the most important pathogenic mechanism influencing the onset and outcome of zygomycosis. Neutrophils cause a lesion of the fungal wall with subsequent destruction by macrophages. They also enhance the activity of antifungal drugs against Zygomycetes. Strategies that aim to increase neutrophil count and function, such as granulocyte colony stimulating factor and granulocyte transfusion, could play an important role in the management of this life-threatening infectious complication.

Isolation and identification of Rhizomucor pusillus from pleural zygomycosis in an immunocompetent patient.

Zygomycosis is usually an invasive mycotic disease caused by fungi in the class Zygomycetes. It often occurs in immunocompromised patients, but sporadic cases without apparent immune impairment have been described. This report presents the first case of pleural zygomycosis caused by Rhizomucor pusillus, an uncommon pathogen of human infection. A 19-year-old man was found to have pleuritis several days after a drainage catheter was implanted to cure a pneumothorax caused by a ruptured bulla. Local pneumonectomy to resect the ruptured bulla and vacuuming of the pleural fluid was performed. Rhizomucor pusillus was cultured from the pleural fluid and irregular broad sparsely septate hyphae, consistent with zygomycetes, were histologically detected in the thickened pleura of the resected bulla. The catheter was suspected of having been contaminated with the fungus, but no evidence could be obtained. His fungal pleuritis subsided without any antifungal medical therapy and his immunocompetence seemed to contribute to limiting the infection.

Vesicular thick-walled swollen hyphae in pulmonary zygomycosis.

An autopsy case of pulmonary zygomycosis in a patient with rheumatoid arthritis on immunosuppressive therapy is presented herein. There was a pulmonary cavitated infarct caused by mycotic thrombosis. Thin-walled narrow hyphae and vesicular thick-walled swollen hyphae were found on the pleural surface and in the necrotic tissue at the periphery of the cavity. Findings of such shaped fungal elements may cause erroneous histopathological diagnosis because pauciseptate broad thin-walled hyphae are usually the only detectable fungal elements in zygomycosis tissue. Although immunohistochemistry confirmed these unusual elements to be zygomycetous in the present case, it is important for the differential diagnosis to be aware that zygomycetes can form thin narrow hyphae and vesicular thick-walled swollen hyphae.

An unusual case of fungal chorioretinitis in an immunocompetent individual.

PURPOSE: To describe a case of fungal chorioretinitis in a healthy individual. DESIGN: Case report. RESULTS: A 74-year-old immunocompetent man presented with peripheral chorioretinitis with hemorrhage in the superonasal quadrant. A retinal biopsy was performed. PAS and Gomori methenamine silver stain disclosed fungal structures identified as broad nonseptate hyphae with right-angle branches, probably belonging to the Zygomycete class. A systemic workup was initiated to seek the source of the infection with normal findings. The patient was successfully treated with intravitreous and intravenous amphotericine B. CONCLUSION: Nontraumatic fungal chorioretinitis can present in immunocompetent patients.

Drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis.

Zygomycosis is an emerging frequently fatal opportunistic mycosis whose immunopathogenesis is poorly understood. We developed a zygomycosis model by injecting Drosophila melanogaster flies with a standardized amount of fungal spores from clinical Zygomycetes isolates to study virulence and host defense mechanisms. We found that, as opposed to most other fungi, which are nonpathogenic in D. melanogaster (e.g., Aspergillus fumigatus), Zygomycetes rapidly infect and kill wild-type flies. Toll-deficient flies exhibited increased susceptibility to Zygomycetes, whereas constitutive overexpression of the antifungal peptide Drosomycin in transgenic flies partially restored resistance to zygomycosis. D. melanogaster Schneider 2 phagocytic cells displayed decreased phagocytosis and caused less hyphal damage to Zygomycetes compared with that to A. fumigatus. Furthermore, phagocytosis-defective eater mutant flies displayed increased susceptibility to Zygomycetes infection. Classic enhancers of Zygomycetes virulence in humans, such as corticosteroids, increased iron supply, and iron availability through treatment with deferoxamine dramatically increased Zygomycetes pathogenicity in our model. In contrast, iron starvation induced by treatment with the iron chelator deferasirox significantly protected flies infected with Zygomycetes. Whole-genome expression profiling in wild-type flies after infection with Zygomycetes vs. A. fumigatus identified genes selectively down-regulated by Zygomycetes, which act in pathogen recognition, immune defense, stress response, detoxification, steroid metabolism, or tissue repair or have unknown functions. Our results provide insights into the factors that mediate host-pathogen interactions in zygomycosis and establish D. melanogaster as a promising model to study this important mycosis.