Evaluation of the vital staining method for <emph type="2">Lacazia loboi</emph> through the experimental inoculation of BALB/c mice

Abstract

The viability of the currently unculturable fungal pathogen Lacazia loboi can be determined by means of fluorescein diacetate-ethidium bromide (FD-EB) staining. This technique can be used in experimental study of the mycosis, in attempts to cultivate the fungus and in attempts to gauge the success of therapies. In the present study, the potential applications of FD-EB vital staining were studied using a proposed murine experimental model of lobomycosis. BALB/c mice were inoculated in the footpads with an L. loboi suspension that appeared in FD-EB staining to have lost viability after being held for 15 days at room temperature, whereas a control group of mice was inoculated with apparently viable fungi. The animals were killed after 1, 2, 4, 6, 8, 11 and 13 months. Both inoculated footpads were excised, one for determination of viability and the other for histological examination. In the group injected with nonviable material, no active infection was noted; inoculation sites showed small quantities of macrophage-laden infiltrate and no viable fungal cells. In the control group, the infection progressed with exuberant infiltrates surrounding copious fungal growth, most of which consisted of cells staining as viable in FD-EB. These results suggest that the FD-EB vital staining is a sensitive and specific method that can reliably be used for viability determination in L. loboi.

Lobomycosis Successfully Treated with Posaconazole

“…This method uses vital staining with fluorescein diacetate-ethidium bromide and has showed consistent sensitivity and specificity for detecting viability in a murine experimental model of lobomycosis. 13 If the good results observed in this animal model are further corroborated in humans, *Address correspondence to Beatriz Bustamante, Av. Honorio Delgado 430, Lima 31, Peru.…”

Lobomycosis: an emerging disease in humans and delphinidae

“…Both in rodents and turtles, the organisms reveal a low number of bizarre forms 104–108 whereas in armadillos the parasite forms and the histological reaction seem almost identical to those seen in humans, a similar phenomenon found also in Mycobacterium leprae inoculations 106 . Recently, with the achievement of new experimental inoculation models, 105–107 it has been possible to develop a sensitive and specific method to determine the tissue viability of Lacazia loboi through the use of vital stains using FD‐EB 109 . Also, through fluorescence, fresh tissue calcofluor white stain it is possible to clearly depict the typical morphological characteristics of the agent 22,105 …”

“…106 Recently, with the achievement of new experimental inoculation models, [105][106][107] it has been possible to develop a sensitive and specific method to determine the tissue viability of Lacazia loboi through the use of vital stains using FD-EB. 109 Also, through fluorescence, fresh tissue calcofluor white stain it is possible to clearly depict the typical morphological characteristics of the agent. 22,105 Treatment Until now, no antifungal has shown to be effective against Lacazia loboi.…”

Análise comparativa do perfil citocínico Th22 na imunopatogenia da doença de Jorge Lobo e cromomicose

“…É considerada uma micose negligenciada sendo causada por um fungo patogênico obrigatório chamado de Lacazia loboi e manifesta-se clinicamente por lesões de aspecto queloidiano que podem acometer qualquer área do tegumento, mas incidem principalmente nos membros inferiores, pavilhão auricular e membros superiores. Predomina entre os países da região amazônica, sobretudo a brasileira, com escassos relatos na Europa, Estados Unidos e Canadá (Opromolla et al, 2003;Marcos et al, 2005;Florian et al, 2020, Brito et al, 2022. -Toro, 1989;Pradinaud, 1998).…”

“…As expressões clínicas em ambas as doenças variam de acordo com a resposta imunológica do paciente, e os mecanismos de defesa influenciam no surgimento e na gravidade dessas infecções fúngicas. Apesar de a resposta imunológica do hospedeiro nessas micoses subcutâneas ser de extrema importância, grande parte dos trabalhos publicados aborda a evolução dessas infecções, baseada, sobretudo, no padrão clássico de expressão de citocinas, sendo poucos os relatos bibliográficos que abordam o papel de novos perfis citocínicos e sua correlação com a resposta imune ao agente infeccioso, bem como sua relação com a evolução da doença (Opromolla et al, 2003;Breda et al, 2021).…”