High-resolution melting analysis assay for identification of Fonsecaea species.

BACKGROUND: Chromoblastomycosis (CBM) is a chronic fungal disease. In China, the principle etiologic agent was a group of dematiaceous fungi, including Fonsecaea monophora, Fonsecaea nubica, and Cladophialophora carrionii. Although the Fonsecaea species have similar morphology, their pathogenicity is quite different. This study aims to establish a new solution for early identification of Fonsecaea species because of their distinctive potential infection risk. METHODS: Five reference strains and 35 clinical isolates from patients with CBM, preserved in our laboratory, were used in this study. The universal primer ITS1 and ITS2 were chosen to amplify the highly conserved regions of rDNA. High-resolution melting (HRM) analysis was performed using the LIGHTCYCLER® 96 System. All the amplicons were verified by direct sequencing and the sequence were aligned with those in GenBank by BLAST analysis. RESULTS: We successfully differentiated the five strains according to their different Tm values and curve shapes. The 35 clinical isolates from patients were identified as 24 strains for F. monophora and 11 strains for F. nubica, which is consistent with the DNA sequencing results. CONCLUSION: It is the first time to use HRM analysis for identification of Fonsecaea species. Since the CBM etiologic agent in South China is mainly F. monophora and F. nubica, this strategy is sufficient to be applied in the clinical examination with high accuracy, speed, and throughput.

Photodynamic effects on Fonsecaea monophora conidia and RAW264.7 in vitro.

Chromoblastomycosis (CBM), one of the neglected tropical diseases, is hard to cure and easy to be recurrent. Many studies suggest that macrophage is involved in the pathogenesis of chromoblastomycosis and the fungicidal effect of 5-Aminolaevulinic Acid-Based Photodynamic Therapy (ALA-PDT) against F. monophora (one of the main causative agent of chromoblastomycosis) has shown great promise. However, the fungicidal ability of ALA-PDT to F. monophora is still controversial and the molecular mechanism and immune mechanism of ALA-PDT against F. monophora remains poorly documented. In the present work, ALA (5-Aminolaevulinic Acid) was employed as photosensitizer and a LED device was served as light source to investigate photodynamic effect on F. monophora conidia under different ALA-PDT conditions in a direct way. RAW264.7 was stimulated by conidia treated with ALA-PDT to study the photodynamic effect on F. monophora conidia in an indirect way. It was observed that ALA-PDT can inactivate F. monophora conidia directly in a concentration-dependent and dose-dependent manner. RAW264.7 was activated indirectly by photodynamically treated conidia. ALA-PDT can enhance the fungicidal ability of RAW264.7 and protect it from Infection-induced apoptosis in an indirect way. ROS generated by photodynamic treated conidia is associated with mitochondrial-related apoptosis in RAW264.7.The results of this investigation demonstrated that ALA-PDT inactivate F. monophora through two way: directly killing F. monophora conidia through ROS-dependent Oxidative damage; activating RAW264.7 in an indirect way.

Molecular identification of chromoblastomycosis clinical isolates in Guangdong.

Chromoblastomycosis is a chronic fungal infection of the skin and subcutaneous tissue. The most common etiologic agent encountered in Southern China is from the genus Fonsecaea. Fonsecaea species are often misidentified due to indistinct morphology features; furthermore, recent taxonomy revision was done on the fungi genus. Herein, a comprehensive evaluation with molecular sequencing data based on internal transcribed spacer (ITS) ribosomal DNA regions as molecular targets were implemented to 37 clinical isolates from chromoblastomycosis patients. Twenty strains that were formerly identified as Fonsecaea pedrosoi through morphological characteristic were verified to be either Fonsecaea nubica or Fonsecaea monophora, while 17 strains were appropriately identified as F. monophora. A phylogenetic method was further performed to establish the species delimitation. Our investigations validate that the clinical isolates from Guangdong consist of F. monophora and the recently found new species, F. nubica. In this study, F. pedrosoi has not been isolated from chromoblastomycosis patients in Guangdong, Southern China. Reevaluation of previous reports regarding F. pedrosoi as chromoblastomycosis etiologic agent in China is necessary for a comprehensive assessment of geographic distribution pattern of Fonsecaea species. This study is the first reported study presenting large samples of F. nubica domestic or abroad.