Microsatellite Typing of Aspergillus Fumigatus

Microsatellite typing of Aspergillus fumigatus isolates recovered from deep organ samples of patients with invasive aspergillosis

Keywords: Invasive Aspergillosis; Aspergillus fumigatus; microsatellite typing; epidemiology

Lies M.E. Vanhee1, Francoise Symoens2, Hans J. Nelis1, Tom Coenye1*

1 Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium

2 Scientific Institute of Public Health, Section of Mycology, Juliette Wytsmanstreet 14, 1050 Brussels, Belgium

Corresponding author: Tom Coenye

Mailing address: Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.

Phone: (32) 9 264 8141, Fax: (32) 9 264 8195.

E-mail:

Abstract

Microsatellite typing was used to analyse 41 Aspergillus fumigatus isolates from nine patients with proven invasive aspergillosis hospitalized in two different centers. No strains were shared between patients. For 8/9 patients, a single genotype was found for the isolates recovered from all anatomical sites involved.

Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus is an often lethal fungal infection, especially in immunocompromised patients (Denning, 1998). The mortality of untreated IA is approximately 85% but it may be reduced to 50% with early treatment (Denning, 1998). In order to develop optimal infection control measures, more insight in the epidemiology of the disease is necessary. Although several typing methods such as RAPD, SSDP and MLEE are available, most of these methods lack the discriminatory power and the reproducibility necessary to distinguish between closely related isolates (Varga, 2006). Bart-Delabesse et al. (1998, 1999) evaluated the use of microsatellite markers and found that this technique has a high discriminatory power for typing Aspergillus fumigatus isolates. In the present study, multiple isolates recovered from various organs of IA patients were typed with a recently developed microsatellite-based method.

Samples from various organs were collected from nine patients (with proven IA) hospitalized in two European medical centers and plated on yeast-extract-peptone-dextrose-chloramphenicol agar or Sabouraud-dextrose-chloramphenicol agar. Strains for this study were isolated during the activities of the EBGA network (Concerted Action Biomed 2 BMH4 97-2481) by J. Meis and P.E. Verweij (Department of Medical Microbiology, University of Nijmegen) and P. Rath (Institut für Medizinische Mikrobiologie, University of Essen). Isolates were identified as A. fumigatus according to standard morphological criteria and their ability to grow at 48°C. All strains were preserved and referenced in the BCCM/IHEM Culture Collection of the Scientific Institute of Public Health (Brussels, Belgium). An overview of the isolates included in the present study is given in Table 1.

Genomic DNA was prepared as described previously (Symoens et al., 2000) and a microsatellite assay was used to genotype all isolates. Nine short tandem repeat loci were amplified using three multiplex PCRs, as described previously (de Valk et al., 2005). PCR products were combined with the ROX400-HD size standard (Applied Biosystems) and analysed by capillary electrophoresis on an ABI3130xl sequencer (Applied Biosystems). Subsequently, Peakscanner (Applied Biosystems) was used to determine the exact size of each amplified fragment and accordingly a locus type was assigned. Combination of these types resulted in an overall genotype (designated by an arbitrary number) for each isolate. A dendrogram was made with START2 softwareusing the unweighted pair group method with arithmetic averages (UPGMA) algorithm.

Microsatellite typing of the 41 isolates from nine patients resulted in the identification of 11 distinct genotypes (Table 1, Fig. 1). A. fumigatus strains were not shared between patients and the genotypes, identified for isolates from individual patients, differed strongly from each other ( 4 loci were different) (Fig. 1). Isolates recovered from patients hospitalized in the same center were not more similar to each other than to isolates recovered from patients hospitalized in the other center (Fig. 1). This is not surprising considering the lack of a clear epidemiological link between patients, even between those hospitalized in the same center. For 8/9 patients, a single strain was found per patient (Table 1). However, for patient ES/1, three genotypes (75, 76 and 82) were identified among the four isolates recovered from various organs. Genotypes 75 and 76 differed at two loci only, while genotype 82 differed at six and seven loci from genotypes 75 and 76, respectively (Fig. 1). These observations suggest that this patient harbored multiple isolates capable of causing invasive disease, although in the case of genotypes 75 and 76, micro-evolution can not be ruled out.

Isolates from IA patients were previously typed by de Valk et al., 2007 and Girardin et al., 1994 using various methods. These studies found that strains were not shared between patients, while within a given patient all isolates recovered from deep organs had the same genotype. In contrast, typing of A. fumigatus isolates recovered from respiratory samples indicated the presence of multiple genotypes (Bertout et al., 2001 and Symoens et al., 2001). This suggests that the respiratory tract is colonised with multiple, distinct A. fumigatus strains but that further invasive infection occurs in the majority of cases with a single one.

Acknowledgements

This research was financially supported by the Belgian Science Policy (contract C3/00/19), the BOF of Ghent University and FWO-Vlaanderen.

The authors would like to thank Jean-Philippe Bouchara, Renée Grillot and Claudine Pinel for stimulating discussions.

References

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Bart-Delabesse E, Cordonnier C, Bretagne S (1999) Usefulness of genotyping with microsatellit markers to investigate hospital-acquired invasive aspergillosis. J Hosp Infect 42: 321-327.

Bertout S, Renaud F, Barton R, Symoens F, Burnod J, Piens M-A, Lebeau B, Viviani, M-A, Chapuis F, Bastide J-M, Grillot R, Mallié M, EBGA network (2001) Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods. J Clin Microbiol 39: 1731-1737.

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Figure Legend

Fig. 1: UPGMA dendrogram of the studied A. fumigatus isolates with center and patient specification (Scalebar indicates a difference of two loci)

Table 1: A. fumigatus isolates included in the present study

IHEM no. / Centera/Patient / Isolation date / Origin of the sample / Microsatellite genotype
15653 / ES/1 / 10-05-1995 / heart b / 75
15654 / ES/1 / 10-05-1995 / right kidney b / 76
15655 / ES/1 / 10-05-1995 / left kidney b / 82
15656 / ES/1 / 10-05-1995 / liver b / 82
15661 / ES/2 / 23-08-1995 / brain b / 80
15662 / ES/2 / 23-08-1995 / heart b / 80
15663 / ES/2 / 23-08-1995 / gallbladder b / 80
15667 / ES/3 / 23-01-1997 / faeces / 83
15668 / ES/3 / 27-01-1997 / throat / 83
15669 / ES/3 / 29-01-1997 / skin / 83
15670 / ES/3 / 03-02-1997 / throat / 83
15671 / ES/3 / 18-02-1997 / faeces / 83
15672 / ES/3 / 20-02-1997 / nose / 83
15673 / ES/3 / 04-03-1997 / nose / 83
15674 / ES/3 / 19-03-1997 / lung b / 83
15675 / ES/3 / 19-03-1997 / heart b / 83
15263 / NI/4 / 01-06-1992 / bronchoalveolar lavage / 73
15264 / NI/4 / 05-06-1992 / right lung b / 73
15265 / NI/4 / 05-06-1992 / left kidney b / 73
15266 / NI/4 / 05-06-1992 / left lung b / 73
15267 / NI/5 / 14-09-1992 / lung b / 74
15268 / NI/5 / 14-09-1992 / brain b / 74
15273 / NI/6 / 03-09-1992 / brain b / 72
15274 / NI/6 / 03-09-1992 / liver abces b / 72
15275 / NI/6 / 03-09-1992 / spleen b / 72
15290 / NI/7 / 04-09-1995 / sputum / 79
15291 / NI/7 / 29-08-1995 / max. sinus aspirate / 79
15292 / NI/7 / 31-08-1995 / faeces / 79
15293 / NI/7 / 14-09-1995 / faeces / 79
15297 / NI/8 / 29-03-1998 / faeces / 62
15298 / NI/8 / 01-04-1998 / left lung b / 62
15299 / NI/8 / 01-04-1998 / right kidney b / 62
15300 / NI/8 / 01-04-1998 / myocard b / 62
15301 / NI/8 / 01-04-1998 / left kidney b / 62
15302 / NI/8 / 01-04-1998 / right lung b / 62
15303 / NI/8 / 01-04-1998 / heart b / 62
15304 / NI/8 / 01-04-1998 / intestine b / 62
15313 / NI/9 / 11-12-1997 / bronchoalveolar lavage / 81
15314 / NI/9 / 17-12-1997 / right lung b / 81
15315 / NI/9 / 17-12-1997 / spleen b / 81
15316 / NI/9 / 17-12-1997 / thrombus heart b / 81

a: ES = Essen, Germany; NI = Nijmegen, The Netherlands

b: These samples were obtained at autopsy. The isolation day of the autopsy samples corresponds to the date of death for all patients. Patient seven from Nijmegen died in October 1995.