A New Aspergillus fumigatus Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP).

Rocio Garcia-Rubio,Emilia Mellado,Teresa Pelaez,Horacio Gil,Maria Candida Monteiro,Gustavo Henrique Goldman

doi:10.1371/journal.pone.0163869

Abstract

Aspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson’s index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks.

Highlights

Aspergillus species are ubiquitous fungi which release huge amounts of spores into the air
Many different molecular methods have been described for typing A. fumigatus, such as random amplified polymorphic DNA (RAPD) [3], amplified fragment length polymorphism analysis (AFLP) [4], restriction fragment length polymorphism analysis (RFLP) [5], and microsatellite length polymorphism (MLP) [6, 7]
Various methodological difficulties have encouraged the development of novel typing techniques, up to date they have shown a lower discriminatory power compare with STRAf

Summary

Introduction

Aspergillus species are ubiquitous fungi which release huge amounts of spores into the air. Aspergillus fumigatus is the leading etiological agent of allergic and bronchopulmonary mycoses and is the most common species causing invasive aspergillosis in immunocompromised. TRESP Genotyping: A New A. fumigatus Typing Method. Investigacion Cooperativa, Ministerio de Economıa y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD 12/0015). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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