Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus

Abstract

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.

Environmental surveillance and other control measures in the prevention of nosocomial fungal infections

“…fumigatus strains [4]. Although most researchers have used the PCR-and RFLP-based typing methods separately, studies are under way to compare their d~scriminatory potential and to evaluate if combination of data obtained by more than one typing method will lead to better strain discrimination [4,33]. To date, strain typing has been most successful by microsatellite polymorphism or analysis of Southern hybridization patterns obtained with repeated DNA sequences [4].…”

Molecular typing of Aspergillus species

“…This probe, called Afut1, possesses specific characteristics of a retrotransposon-like element 33 and has been used in most RFLP studies so far. 6,14,34 Other probes used for fingerprinting of A. fumigatus, which are based on retrotransposon-like elements are Afut2 35 and Af4A. 36 McAlphin et al constructed and characterised a species-specific probe for A. flavus named pAF28.…”

“…74 In contrast, RFLP either with or without hybridisation, as non-PCR-based methods, need relatively large amounts of highly purified DNA which make them time-consuming and labour-intensive. 6,34 Several PCR-based methods like AFLP, CSP, MLST, microsatellites, RISC and RAMS need special equipment to separate obtained fragments with high resolution electrophoresis conditions. On the other hand the RAPD method can be performed with relatively inexpensive equipment and is therefore the most widely used method for typing Aspergillus isolates.…”

“…5 Of all aspergilli, Aspergillus fumigatus is the organism most frequently isolated from human infections. 6 Aspergillus flavus is the second most common aetiological agent of human aspergillosis. 7 Besides these two, other aspergilli like Aspergillus niger, Aspergillus nidulans and Aspergillus terreus can also be the causative agents of various forms of infections.…”

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus

“…To better distinguish between similar strains, the genetic relatedness of clinical isolates of A. fumigatus was determined by a multiplex approach. RAPD analysis involving the independent probe R108, which discriminates between A. fumigatus isolates [11,14], was combined with classical RFLP type mapping utilizing a 1.1-kb hybridization probe recognizing a retrotransposon-like element from A. fumi-gatus, Afut1 [5,16] and the markers Afd and Af4A. Clinical isolates from four patients colonized with A. fumigatus were chosen to examine potential strain di¡erences.…”