Combined used of RAPD and touchdown PCR for epidemiological studies of Aspergillus fumigatus

• Published in: Enfermedades infecciosas y microbiología clínica Vol. 21; no. 9; pp. 472 - 476
• Main Authors: Treviño-Castellano, Mercedes, Rodríguez-Nóvoa, Sonia, Llovo-Taboada, José, García-Zabarte, Angeles, García-Riestra, Carlos, Regueiro-García, Benito José
• Format: Journal Article
• Language: English; Spanish
• Published: Spain 01.11.2003
• Subjects: Aspergillosis - diagnosis; Aspergillosis - epidemiology; Aspergillosis - microbiology; Aspergillus fumigatus - classification; Aspergillus fumigatus - genetics; Aspergillus fumigatus - isolation & purification; Cross Infection - diagnosis; Cross Infection - epidemiology; Cross Infection - microbiology; DNA, Fungal - analysis; Genotype; Humans; Mycological Typing Techniques; Opportunistic Infections - diagnosis; Opportunistic Infections - epidemiology; Opportunistic Infections - microbiology; Polymerase Chain Reaction; Random Amplified Polymorphic DNA Technique; Species Specificity; Time Factors
• ISSN: 0213-005X

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. Description of a fast, sensitive, specific method to identify and fingerprint A. fumigatus using methodology available in clinical laboratories. 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. 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. 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.