Utility of a microsatellite assay for identifying clonally related outbreak isolates of Aspergillus fumigatus

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Abstract

A microsatellite assay based on short tandem repeats (STRAf) has been recently described as a discriminatory, high throughput assay for fingerprinting Aspergillus fumigatus isolates. However, the STRAf assay has not been tested for its utility in outbreak settings where it is critical to distinguish clonal clusters from genetically unrelated genotypes. In the present study, employing a panel of epidemiologically linked A. fumigatus isolates obtained from 6 different outbreaks of invasive aspergillosis (IA), we demonstrate that the STRAf assay can be a valuable molecular tool to support epidemiological investigations. We also report for the first time the detection of microvariation events in the A. fumigatus population studied.

Introduction

Invasive aspergillosis (IA) is a life-threatening disease in immunocompromised patient populations and evidence suggests that such infections may be acquired from hospital environments; accordingly, numerous outbreaks of IA, largely due to Aspergillus fumigatus, have been reported (Bretagne et al., 1997, Leenders et al., 1996, Munoz et al., 2004, Pegues et al., 2002). A molecular typing method that can distinguish “clonally related” or genetically related isolates from unrelated isolates can be a powerful tool in outbreak investigations where the assumption is that isolates with identical genotypes (or “clonal” clusters) could indicate a common source of exposure, whereas epidemiologically unrelated isolates will have different genotypes (Singh et al., 2006). Thus, genotyping methods can indicate the source and map the route of transmission of IA, thereby informing epidemiological investigations and ultimately guide prevention strategies.

Numerous molecular methods have been evaluated for sub-typing A. fumigatus including microsatellite based fingerprinting assays. Recently, a robust microsatellite assay, the STRAf assay, based on nine, short tandem repeat loci has been described for high resolution fingerprinting of A. fumigatus (de Valk et al., 2005, de Valk et al., 2007a). Previously, the STRAf assay was able to segregate 99 presumably unrelated A. fumigatus isolates into 96 unique genotypes demonstrating its very high discriminatory power (de Valk et al., 2005), delineate unique genotypes amongst A. fumigatus isolates recovered from multiple different patients in a single hospital, and finally identified genetically related isolates from different body sites of the same patient (de Valk et al., 2007b). Thus, the STRAf assay satisfies a critical measure of a good typing system, that is, it can provide distinctive fingerprints from genetically unrelated isolates and identical or highly similar fingerprints from closely related organisms. According to the guidelines of the European Study Group on Epidemiological markers (ESGEM), another important criterion of an ideal typing scheme is the system's capacity to establish epidemiological concordance among strains recovered from the same outbreak, which can be evaluated by testing sets of isolates from different outbreaks containing multiple isolates per outbreak (van Belkum et al., 2007) . However, it is not known whether the STRAf assay can fulfill this criterion, and the present study was designed to evaluate the STRAf assay for its utility as a molecular tool to support outbreak investigations of aspergillosis.

The Mycotic Diseases Branch at the Centers for Disease Control and Prevention has a collection of several A. fumigatus isolates from multiple outbreak investigations across Northern America (Lasker, 2002, Panackal et al., 2003, Pegues et al., 2002). These isolates have been subjected to several genotyping methods to assess their genetic relatedness to support epidemiological investigations including the Afut1 DNA hybridization profiles (Afut1 method) and a recently developed single locus sequencing typing method employing the putative cell surface protein gene Afu3g08990, designated as “CSP typing” method (Balajee et al., 2007). Given the availability of relevant epidemiological data for each outbreak investigation, this panel was uniquely suited and was therefore chosen to test the suitability of the STRAf method to type epidemiologically linked A. fumigatus isolates.

Section snippets

Aspergillus isolates used in the study

A total of 55 A. fumigatus isolates obtained from six different outbreak investigations (denoted as OB1–OB6) were included in this study.

Isolates in cluster OB1 were obtained from a renal transplant unit in California. This outbreak was thought to be associated with construction related activities; however genotyping results using the Afut1 method showed that the isolates recovered from the environment were unrelated to the clinical isolates from patients (Panackal et al., 2003). Outbreak OB2

Results

Results of the STRAf assay for cluster OB1 showed that 3 clinical isolates 607, 683 and 686 belonged to a clonal cluster while all other isolates representing mostly environmental isolates consisted of unrelated genotypes (Fig. 1A). In cluster OB2, 11 isolates formed a clonal cluster and 1 clinical isolate had an unrelated genotype (Fig. 1B). Here, isolates B5850, B5864, B5852 and 38 recovered from a single patient (whose abdominal wound was implicated as the source of the outbreak), isolate B5856

Discussion

Recently, a method that employs a panel of short repeats from 9 loci in multiplex PCRs where the reaction amplified three di-, tri-, or tetranucleotide repeats respectively (the STRAf assay) has been described for fingerprinting A. fumigatus isolates (de Valk et al., 2005). With this assay, the combination of all nine markers yielded a Simpson's diversity index of 0.9994, indicating that the method had a high discriminatory power. Theoretically this panel of markers can discriminate between up

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