Pythium insidiosum: avaliação de imunoterápicos para eqüinos, utilizando-se coelhos como modelo experimental.

A pitiose é uma doença granulomatosa, tendo como agente etiológico Pythium insidiosum De Cock, 1987, que atinge eqüinos, provocando quadro infeccioso na pele e tecido subcutâneo , caninos com apresentação gastrintestinal e cutânea , bovinos com doença cutânea , felinos e humanos, com quadro clinico de arterite, queratite e celulite periorbital. Esta enfermidade é mais prevalente em áreas tropicais, subtropicais ou temperadas. Também animais silvestres podem se infectar pela doença. O gênero Pythium pertence ao Reino Stramenopila, Filo Oomycota, cujos membros caracterizam-se por produção de zoósporos biflagelados durante a reprodução assexuada. O desenvolvimento de pitiose experimental nas espécies naturalmente infectadas não foi ainda relatado, mas os coelhos são sensíveis à inoculação de zoósporos e podem ser usados como modelo experimental para estudo da pitiose. Este trabalho teve como objetivo avaliar a eficiência de 3 processos de produção de imunoterápicos contra pitiose, produzidos a partir do cultivo e posterior maceração ou sonicação, em coelhos infectados experimentalmente com 17.500 zoósporos do oomiceto Pythium insidiosum (cepa CBS 101555). Todos os coelhos foram inoculados com zoósporos 1 mês antes da aplicação dos imunoterápicos. Para avaliação dos imunoterápicos, os coelhos que desenvolveram lesões foram divididos em 4 grupos de 5 animais: Grupo 1 – Tratado com placebo; grupo 2 – tratado com o imunoterápico sonicado; grupo 3 – tratado com o imunoterápico misto e o grupo 4 - Imunoterápico macerado mecanicamente. Todos os animais receberam 8 doses do imonoterápico ou placebo com intervalos de 14 dias. Um mês após a inoculação dos zoósporos móveis, foram iniciadas as medições das áreas inoculadas. Os resultados indicaram que o imunoterápico macerado, utilizado no grupo 4, foi estatisticamente (P<0.001) mais eficiente que os demais, diminuindo em até 71,8% a área dos nódulos provocados pelo Pythium insidiosum, após 26 semanas de avaliação. Neste período 2 coelhos deste grupo foram curados. Os animais do Grupo 2 que receberam o imunoterápico sonicado, não mostraram nenhuma reação, detectando-se aumento de até 221% no tamanho das lesões. Nos coelhos do grupo 3, imunoterápico misto, houve aumento das lesões em 50%. A provável causa deste insucesso com o grupo 2, está na desnaturação dos antígenos protetores através dos processos de sonicação. Os dados gerados nesta tese podem inferir que, futuramente, novas perspectivas se abrem para o estudo da pitiose e seu controle.

Does Immunotherapy Protect Equines from Reinfection by the Oomycete Pythium insidiosum?

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Pythium insidiosum: relato do primeiro caso de infecção humana no Brasil

A pitiose é causada por microorganismo aquático, fungo-símile, o Pythium insidiosum, patógeno de homens e animais. Observou-se um paciente com úlcera fagedênica no membro inferior, com exame anatomopatológico sugestivo de zigomicose, pouco sensível à terapêutica antifúngica, obtendo-se cura por meio de ampla exérese. A comprovação etiológica resultou de métodos moleculares, com amplificação e seqüenciamento de DNA de organismo isolado em ágar Sabouraud, observando-se 100% de analogia com seqüências de P. insidiosum depositadas no GenBank.

Morphological and molecular characterization of an equine isolate of Pythium insidiosum and comparison with the first human isolate from the same geographic region

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Photodynamic Therapy in Pythium insidiosum - An In Vitro Study of the Correlation of Sensitizer Localization and Cell Death

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do Efeito Fotodinâmico sobre o Crescimento in vitro de Pythium insidiosum

Photodynamic Therapy (PDT) is a therapeutic method which employs a photosensitizer and light to cause cellular death. The chemical compounds have low or none toxicity for hosts cells. Under the incidence of light, in an appropriated wavelength, these chemical compounds produce reactive oxygen which affects the biomolecules of the target-cells. The specific illumination of the affected area increases the selectivity of the therapy, since the photodynamic process occurs only in the irradiated area. Pythiosis, for instance, is a life-threatening emerging disease caused by a fungus-like organism called Pythium insidiosum. The disease occurs in man and other animals, being mostly observed in horses. Human pythiosis may present as ophthalmic, cutaneous-subcutaneous and systemic forms of lesions. Due to the fact that P. insidiosum is not a true fungus, it is refractory to most antifungal drugs and the treatment of the disease is difficult. Extensive surgery procedures, such as limb amputation, are the treatment of choice, however relapses may occur frequently. Although not totally effective, the use of immunotherapy associated to surgery have shown some results. Considering that pythiosis is an emerging disease few explored in its etiological and therapeutic aspects, which are limited and few effective, it is of great importance to encourage the development of researches for new strategies of treatment. In this sense, it was evaluated the effect of PDT on in vitro growth of the pathogen employing two chemical compounds as photosensitizer, porphyrin and chlorine, at different concentrations in combination with several energetic dosages. Porphyrin showed inhibition of growth at 25mg/mL with 100J/cm2 of energetic dosage and chlorine showed similar results employing low concentrations (0,7, 1,0 and 1,3mg/mL) with 70J/cm2 of energetic dosage... (Complete abstract click electronic access below)

Efeito do ácido tânico purificado (tanino) sobre o crescimento in vitro de isolados de Pythium insidiosum

Pythiosis is caused by the oomycetous Pythium insidiosum and affect domestic and wild animals and man. The presence of water and vegetal material is fundamental for its life cycle in nature. The biflagellate zoospore are the infective form of this pathogen. The lesions are generally of granulomatous aspect, which frequently may be contaminated by secondary bacterial infection in skin and subcutaneous tissue. Dissemination to systemic tissues may also occur and it may be due to the spread of the pathogen from cutaneous lesions, as well as a primary source of infection. Clinical signs depend on the affected tissue. Diagnosis of pythiosis is based on the clinical manifestations, histopathological sections and culture of the pathogen. Serological tests may also be employed and more recently molecular biology has been introduced as a sensitive, specific and a rapid method for conclusive diagnosis. Treatment is often difficult and extensive surgery procedures are required, however, depending on the anatomic region and size of the lesion, such procedure is unfeasible and relapses are frequent. Due to the climate changes, which has contributed to increase the incidence of pythiosis, it is necessary the search for new therapeutic protocols

Caracterização do Pythium insidiosum por abordagem proteômica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Pitiose cutânea equina de localização atípica tratada topicamente com solução de anfotericina B e DMSO

Background: Cutaneous lesions by Pythium insidiosum infection are commonly observed in horses, especially in those living at flooded environments. Equine pythiosis is characterized by the development of tumoral masses that are frequently located at distal limbs, ventral abdomen, thorax, breast and face. The lesions are usually granulomatous, serosanguineous and ulcerated, most often destroyed by self-mutilation due to the intense pruritus. The proposed treatment includes surgical excision followed by antifungal drugs administration, which can be done systemically or topically. Amphotericin B and dimethyl sulfoxide (DMSO) in association has been successfully used for cutaneous pythiosis topical treatment due to the DMSO property to carry any substance through plasmatic membranes.Case: The present report concerns a 12-year-old mixed breed gelding presenting with self-mutilation of a tumoral mass located at the left flank. The owners reported that the horse had initially presented a small wound that had evolved to a 20-cm in diameter mass in 4 weeks. Tissue samples were collected, processed and stained by the Gomori's methenamine silver (GMS) method. The histopathological analysis revealed Pythium insidiosum hyphae in a granulomatous tissue, especially located at peripheral region, where kunkers were present. Surgical excision of the mass followed by cauterization was indicated as initial treatment, and due to financial reasons, the owners elected only the topical antifungal therapy to control the fungus infection after surgery. Flunixin meglumine was also administrated for five days aiming the control of pain and inflammation. The wound was cleaned with povidone-iodine solution and rinsed with a solution containing, 50 mg, of amphotericin B in 10 mL of sterile water and 10 mL of DMSO. This procedure was carried Out twice a day. The wound healed fast due to an excellent centripetal epithelialization. and the horse was discharged after 64 days showing only 5% of the initial wound area. The owner reported by telephone the complete healing and hair growth 10 days after discharge.Discussion: Despite the atypical location of the tumoral lesion described at the present report, the history and clinical manifestations, especially the intense pruritus, showed similarity with other characteristic reports of equine cutaneous pythiosis. The diagnosis was confirmed by the histopathological examination showing hyphae structures, as described to be evidences of the presence of Pythium insidiosum in the tissue. The surgical procedure was the first step to provide remission of clinical signs, and one day after surgery the pruritus desapeared. After excision of the granulomatous tissue and cauterization, daily topical administration of amphotericin B associated with DMSO was effective in destroying the infectious agent, as observed by the excellent epithelization. A pink granulation tissue grew up providing an ideal surface for epithelial migration and the healing process progressed quickly. Centripetal epithelialization reduced the wound area until 3% of the initial area in 64 days of treatment, when the remaining wound was found almost completely healed and covered with hair. At the present report, the horse presenting pythiosis was only topically treated. The recommended therapy using amphotericin B and DMSO solution was effective, economically viable and low risk, considering that the systemic antifungal therapy usually suggested is expensive and extremely nephrotoxic. The atypical location of the lesion on the left flank shows that any anatomical region can be affected by the fungus, since the conditions for its development were present.

Cutaneous pythiosis in a dog from Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Pitiose em animais de produção no Pantanal Matogrossense

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Treatment of Pythiosis in Equine Limbs Using Intravenous Regional Perfusion of Amphotericin B

Objective To evaluate the effects of intravenous regional limb perfusion (IRLP) administration of amphotericin B in horses to treat pythiosis after surgical excision and thermocautery. Study Design Case series. Animals Horses (n = 12) with Pythium insidiosum infection of the distal aspect of the thoracic or pelvic limbs. Methods After surgical excision of granulation tissue and thermocautery, 50 mg amphotericin B was administered by IRLP through a catheter placed in a superficial vein of the affected limb next to the lesion after placing a tourniquet above the injection site. The lesions and locomotor system were evaluated before treatment and at 7, 14, 21, 28, 35, and 60 days. Results Ninety-two percent of horses treated with amphotericin B had complete lesion resolution 35 or 60 days after 1 or 2 IRLP treatments, respectively. IRLP induced limb edema and pain during regional palpation in 42%, and inflammation of the injection site in 33% of horses; however these signs resolved after 14 days. Conclusions IRLP administration of amphotericin B was effective for treating pythiosis in equine limbs, resolving infection with manageable side effects.

Human pythiosis, Brazil

Pythiosis, caused by Pythium insidiosum, occurs in humans and animals and is acquired from aquatic environments that harbor the emerging pathogen. Diagnosis is difficult because clinical and histopathologic features are not pathognomonic. We report the first human case of pythiosis from Brazil, diagnosed by using culture and rDNA sequencing.

Pitiose nasal em equino

Pythiosis is a subcutaneous infection caused by aquatic fungi (Pythium insidiosum) which occurs in some animal species, as human beings. This paper describes an Quarter Horse, breed in Pernambuco, Brazil, who had this infection. The horse presented nodular and ulcerated lesions only in nasal mucosa and the diagnosis was made by histopathologic analysis and clinical signals. Based in the histopathologic findings and anamneses information by the owner the authors believed this is an atypical presentation of this infection caused by the contact with water offered at stable.

Photodynamic therapy for pythiosis

Background -  Pythiosis is a life-threatening disease caused by Pythium insidiosum. Photodynamic therapy (PDT) is an alternative treatment to surgery that uses the interaction of a photosensitizer, light and molecular oxygen to cause cell death. Objectives -  To evaluate the effect of PDT on the in vitro growth of P. insidiosum and in an in vivo model of pythiosis. Methods -  For in vitro studies, two photosensitizers were evaluated: a haematoporphyrin derivative (Photogem®) and a chlorine (Photodithazine®). AmphotericinB was also evaluated, and the control group was treated with sterile saline solution. All experiments (PDT, porphyrin, chlorine and light alone, amphotericinB and saline solution) were performed as five replicates. For in vivo studies, six rabbits were inoculated with 20,000 zoospores of P. insidiosum, and an area of 1cm3 was treated using the same sensitizers. The PDT irradiation was performed using a laser emitting at 660nm and a fluence of 200J/cm2. Rabbits were clinically evaluated daily and histopathological analysis was performed 72h after PDT. Results -  For in vitro assays, inhibition rates for PDT ranged from 60 to 100% and showed better results in comparison to amphotericinB. For the in vivo assays, after PDT, histological analysis of lesions showed a lack of infection up to 1cm in depth. Conclusions and clinical importance -  In vitro and in vivo studies showed that PDT was effective in the inactivation of P. insidiosum and may represent a new approach to treating pythiosis. © 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.