Identification of Invasive Filamentous Mold Isolates Using DNA Sequencing: Experience of a Clinical Laboratory in a Resource-limited Setting

Abstract

Background: Correct identification of clinically significant mold is becoming critical and cannot be relied only on phenotypic methods; hence, there is a dire need to develop an algorithm of workflows and capacity for molecular identification. This study shares the experience of DNA sequencing for invasive mold identification isolated and compared with phenotypic identification. Methods: This study was conducted at the microbiology laboratory, Aga Khan University, Karachi, Pakistan, and the Mycotic Disease Branch, Centers for Disease Control and Prevention, USA. Filamentous molds isolated from clinical specimens during January 2012–April 2013 were initially identified through phenotypic characteristics. Pan-fungal polymerase chain reaction targeting the internal transcribed spacer region of the ribosomal cistron and the D1/D2 domains of the 28S ribosomal cistron was performed. Sequencer version was utilized to edit and align the DNA sequences, and then sequences were identified using BLAST. The correlation between phenotypic and molecular identification was evaluated. Results: Gene sequencing identified 50% of clinical isolates as one of the Aspergillus species, followed by Mucorales 29%, Fusarium species 17%, and Cladosporium spp. 4%. Overall, 50% of clinical isolates were identified correctly till the species level by conventional methods. Phenotypic correlation with genotype till genus was 42%, while two isolates were wrongly identified phenotypically. Excellent agreement (100%) till species level between phenotypic and molecular identification for Aspergillus, while Mucorales had an agreement of 71%. Conclusion: Traditional phenotypic identification methods for filamentous molds had a good correlation with sequencing up to genus level identification; however, were not reliable up to species level.