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


CONCLUSIONS
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.
Servicio de Microbiología. Complejo Hospitalario Universitario de Santiago de Compostela (CHUS). A Coruña. España. mecedes.trevino.castellano@sergas.es
Mercedes
M
Treviño-Castellano
Sonia
S
Rodríguez-Nóvoa
José
J
Llovo-Taboada
Angeles
A
García-Zabarte
Carlos
C
García-Riestra
Benito José
BJ
Regueiro-García
D001228:Aspergillosis / Q000175:diagnosis* / Q000453:epidemiology / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D003428:Cross Infection / Q000175:diagnosis* / Q000453:epidemiology / Q000382:microbiology; D004271:DNA, Fungal / Q000032:analysis; D005838:Genotype; D006801:Humans; D016533:Mycological Typing Techniques; D009894:Opportunistic Infections / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology; D016133:Polymerase Chain Reaction; D019105:Random Amplified Polymorphic DNA Technique; D013045:Species Specificity; D013997:Time Factors
D004271:DNA, Fungal