INTRODUCTION: Aspergillus fumigatus is a filamentous fungus that acts as an opportunistic pathogen and has emerged as a major problem in immunosuppressed patients. Nosocomial outbreaks of aspergillosis are becoming more frequent, but their identification and epidemiological characterization is slow and difficult. OBJECTIVE: Description of a fast, sensitive, specific method to identify and fingerprint A. fumigatus using methodology available in clinical laboratories. METHODS: We studied several strains of A. fumigatus isolated from patients with invasive aspergillosis (n = 4), the hospital environment (n = 5) and reference cultures (n = 1), as well as other close phylogenetic fungal species from patients (n = 1), hospital environment (n = 6) and reference cultures (n = 1). A. fumigatus was identified by both touchdown PCR and conventional phenotyping methods. Genotyping was performed with random amplification of polymorphic DNA (RAPD) analysis, comparing the results from two primers (OPZ-19 and R-108) and different amplification protocols with regard to band resolution and reproducibility. RESULTS: Touchdown PCR and phenotype results were identical. Best RAPD results were obtained with the R-108 primer and considerably longer ramp times between annealing and extension. CONCLUSION: RAPD analysis is a fast, reliable tool for DNA fingerprinting. Patterns may be easier to repeat and interpret when longer ramp times are used. Touchdown PCR combined with RAPD analysis is a sensitive, accurate method for managing clinical outbreaks of Aspergillus fumigatus.
INTRODUCTION:Aspergillus fumigatus is a filamentous fungus that acts as an opportunistic pathogen and has emerged as a major problem in immunosuppressed patients. Nosocomial outbreaks of aspergillosis are becoming more frequent, but their identification and epidemiological characterization is slow and difficult. OBJECTIVE: Description of a fast, sensitive, specific method to identify and fingerprint A. fumigatus using methodology available in clinical laboratories. METHODS: We studied several strains of A. fumigatus isolated from patients with invasive aspergillosis (n = 4), the hospital environment (n = 5) and reference cultures (n = 1), as well as other close phylogenetic fungal species from patients (n = 1), hospital environment (n = 6) and reference cultures (n = 1). A. fumigatus was identified by both touchdown PCR and conventional phenotyping methods. Genotyping was performed with random amplification of polymorphic DNA (RAPD) analysis, comparing the results from two primers (OPZ-19 and R-108) and different amplification protocols with regard to band resolution and reproducibility. RESULTS: Touchdown PCR and phenotype results were identical. Best RAPD results were obtained with the R-108 primer and considerably longer ramp times between annealing and extension. CONCLUSION: RAPD analysis is a fast, reliable tool for DNA fingerprinting. Patterns may be easier to repeat and interpret when longer ramp times are used. Touchdown PCR combined with RAPD analysis is a sensitive, accurate method for managing clinical outbreaks of Aspergillus fumigatus.
Authors: Omar E Valencia-Ledezma; Carlos A Castro-Fuentes; Esperanza Duarte-Escalante; María Guadalupe Frías-De-León; María Del Rocío Reyes-Montes Journal: J Fungi (Basel) Date: 2022-03-13