Evaluation of Performance of Four Genotypic Methods for Studying the Genetic Epidemiology of Aspergillus fumigatus Isolates

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.

Use of a Novel Panel of Nine Short Tandem Repeats for Exact and High-Resolution Fingerprinting of Aspergillus fumigatus Isolates

“…However, our results may indicate that the three profiles found twice were actually from related isolates. Unfortunately, studies comparing different fingerprinting techniques for A. fumigatus show that, up to now, no single technique can be considered the "gold standard" (3,8). Additional fingerprinting of these isolates by AFLP analysis (18) also showed that these three pairs of isolates were indistinguishable from each other (results not shown).…”

“…Genotypic methods include several techniques, such as random amplified polymorphic DNA (RAPD) analysis (1,10,11), restriction fragment length polymorphism (RFLP) analysis (13), amplified fragment length polymorphism (AFLP) analysis (18), and microsatellite length polymorphism (2). The last technique is sometimes also called microsatellite polymorphism analysis (5) or polymorphic microsatellites marker analysis (8). However, a major problem with pattern-based techniques, such as RAPD, RFLP, and AFLP analyses, is the poor interlaboratory reproducibility.…”

Molecular Epidemiology of Aspergillus fumigatus Isolates Recovered from Water, Air, and Patients Shows Two Clusters of Genetically Distinct Strains

“…We used AFLP fingerprinting for determining the epidemiologic relatedness between the collected isolates. To date several other PCR fingerprinting methods have been described for A. fumigatus, but they either lack the necessary reproducibility between experiments (like Random Amplified Polymorphic DNA analysis [RAPD]) or require previous genomic information (like microsatellite analysis) (14). AFLP finger- printing, however, has proven to be a generic fingerprinting method with intrinsic superior reproducibility and a high degree of discriminatory power compared to other random amplification methods like RAPD (19).…”

Population structure and diversity of the pathogenic fungus Aspergillus fumigatus isolated from different sources and geographic origins

“…Although numerous papers were published recently on the application of several typing techniques to A. fumigatus isolates, only some authors attempted to examine the reproductive mode of A. fumigatus populations and the conclusions drawn by them were controversial (Girardin et al 1994, Fridkin & Jarvis 1996, Lasker 2002, Levdansky et al 2007. In Latin America, there is very little information on these different aspects of the epidemiology of aspergillosis.…”