Comparison of High-resolution Melting Curve Analysis with Specific Target Gene Sequencing to Identify the Most Common Species of Aspergillus and Fusarium

Abstract

Background: Currently, it appears that new molecular-based methods could substitute microscopic and culture assessment for the first-line detection of microorganisms isolated from clinical specimens. However, it will remain the "continual strategy" until this technology is attuned to identifying all fungi that can be isolated from biological specimens. Objectives: The present study aimed to validate a high-resolution melting (HRM) technique to identify clinical filamentous fungi. Moreover, it was attempted to compare the results with those of the target gene’s polymerase chain reaction (PCR) sequencing. Methods: A total of 54 specimens of bronchoalveolar lavage (BAL), nail, ear discharge, blood culture, and cornea were collected from patients suspected of fungal infection. All Fusarium spp. and Aspergillus spp. were recognized based on Tef-α and beta-tubulin region sequencing, as well as PCR-HRM analysis. Results: The Tef-α sequence analysis revealed the most frequent spp. to be Fusarium solani followed by F. oxysporum (n = 3), F. caucasicum (n = 3), F. coeruleum (n = 3), F. falciforme (n = 1), F. proliferatum (n = 1), F. brevicatenulatum (n = 1), F. globosom (n = 1), and F. verticillioides (n = 1). Based on the beta-tubulin sequences, Aspergillus flavus (n = 10), A. fumigatus (n = 7), A. niger (n = 2), A. terreus (n = 1), and A. orezea (n = 1) were identified in this study. Furthermore, the dataset analysis of PCR-HRM revealed that 33 isolates belonging to Fusarium spp. were F. solani (n = 24), F. oxysporum (n = 3), F. proliferatum (n = 3), F. falciforme (n = 1), F. verticillioides (n = 1), and F. brevicatenulatum (n = 1). Moreover, isolates (n = 21) belonging to Aspergillus spp. included A. flavus (n = 11), A. fumigatus (n = 7), A. niger (n = 2), and A. terreus (n = 1). Conclusions: The sequencing method has a time-consuming and costly nature, and there exists conformity between the sequence results of the Tef-α/beta-tubulin regions and PCR-HRM. The PCR-HRM method is a reliable approach in the clinical laboratory to identify Aspergillus and Fusarium spp. However, some closely related spp. show no curve algorithm differences in PCR-HRM.