Abstract
In the present investigation, 49 Aspergillus fumigatus isolates obtained from four nosocomial outbreaks were typed by Afut1 restriction fragment length polymorphism (RFLP) analysis and three PCR-based molecular typing methods: random amplified polymorphic DNA (RAPD) analysis, sequence-specific DNA primer (SSDP) analysis, and polymorphic microsatellite markers (PMM) analysis. The typing methods were evaluated with respect to discriminatory power (D), reproducibility, typeability, ease of use, and ease of interpretation to determine their performance and utility for outbreak and surveillance investigations. Afut1 RFLP analysis detected 40 types. Thirty types were observed by RAPD analysis. PMM analysis detected 39 allelic types, but SSDP analysis detected only 14 types. All four methods demonstrated 100% typeability. PMM and RFLP analyses had comparable high degrees of discriminatory power (D = 0.989 and 0.988, respectively). The discriminatory power of RAPD analysis was slightly lower (D = 0.971), whereas SSDP analysis had the lowest discriminatory power (D = 0.889). Overall, SSDP analysis was the easiest method to interpret and perform. The profiles obtained by PMM analysis were easier to interpret than those obtained by RFLP or RAPD analysis. Bands that differed in staining intensity or that were of low intensity were observed by RAPD analysis, making interpretation more difficult. The reproducibilities with repeated runs of the same DNA preparation or with different DNA preparations of the same strain were high for all the methods. A high degree of genetic variation was observed in the test population, but isolates were not always similarly divided by each method. Interpretation of band profiles requires understanding of the molecular mechanisms responsible for genetic alternations. PMM analysis and Afut1 RFLP analysis, or their combination, appear to provide the best overall discriminatory power, reproducibility, ease of interpretation, and ease of use. This investigation will aid in planning epidemiologic and surveillance studies of A. fumigatus.
Highlights
49 Aspergillus fumigatus isolates obtained from four nosocomial outbreaks were typed by Afut1 restriction fragment length polymorphism (RFLP) analysis and three PCR-based molecular typing methods: random amplified polymorphic DNA (RAPD) analysis, sequence-specific DNA primer (SSDP)
Afut1 RFLP analysis, SSDP analysis, and polymorphic microsatellite markers (PMM) analysis were species specific; amplification products were observed by RAPD analysis for non-A. fumigatus species of Aspergillus
Afut1 RFLP and PMM analyses had comparable high degrees of discriminatory power (D ⫽ 0.989 and 0.988, respectively) (Table 2). These estimates for the discriminatory power of PMM analysis are in good agreement with those from the investigation by Bart-Delabesse et al [4] for a collection of 102 isolates obtained from three Paris hospitals (D ⫽ 0.994)
Summary
(18), except that Afut DNA was labeled with digoxigenin by random priming and hybridization bands were visualized with the reagents and by the protocol supplied in the Genius kit (Roche Diagnostics Corp.). Isolates were assigned a different type by Afut RFLP analysis when two or more band differences were observed between hybridization profiles [5, 30]. Primers R108, R151, and UBC90 for RAPD analysis, the conditions for PCR amplification, reagents, agarose gel electrophoresis, and inspection and interpretation of ethidium bromide-stained gels were as described by Lin et al [25]. The five pairs of primers for SSDP analysis, the PCR amplification conditions, reagents, resolution of the amplified PCR products by agarose gel electrophoresis, and genotyping were as described previously by Mondon et al [27], except that AmpliTaq DNA polymerase PCR amplification was performed with Taq DNA polymerase (Roche Diagnostics Corp.) in a GeneAmp
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