High-resolution melting analysis assay for identification of Fonsecaea species

Abstract

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.

Keywords: Fonsecaea species; chromoblastomycosis; high-resolution melting; identification.

Figure 1

Multiple‐sequence alignment by Lasergene MegAlign

Figure 2

Results of HRM analysis. All the strains were analyzed in triplicate and are specified with lines on the figure. One color represents one strain. (A) Normalized melting peaks of the five strains. All curves show two peaks, which are corresponding to the Tm1 and Tm2 values. (B) Difference plot of the five strains. Take F. nubica as baseline sample, the curves are divided into three groups: C. carrionii, E. dermatitidis, and Fonsecaea species. (C) Normalized melting peaks of the Fonsecaea species. Although the Tm values of them are similar, the three Fonsecaea species can be identified thanks to the small hump after the second peak. (D) Difference plot of the Fonsecaea species. Take F. nubica as baseline sample, the distinction among the three Fonsecaea species is more obvious visually