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Journal of Clinical Microbiology, July 2001, p. 2683-2686, Vol. 39, No. 7
Laboratoire de Parasitologie-Mycologie, Hôpital Henri
Mondor, Créteil,1 and Unité
des Aspergillus, Institut Pasteur, Paris,2
France, and Department of Medical Microbiology and Infectious
Diseases, Erasmus Medical Center, Rotterdam, The
Netherlands3
Received 21 December 2000/Returned for modification 18 March
2001/Accepted 24 April 2001
Aspergillus fumigatus fingerprints generated by
random amplification of polymorphic DNA (RAPD), restriction fragment
length polymorphism (RFLP) upon hybridization with repeated DNA
sequences, and PCR detection of microsatellite length polymorphism
(MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.
Aspergillus fumigatus is
the main species responsible for invasive aspergillosis, an often-fatal
infection occurring in immunocompromised patients (27). In
nature, this fungal species thrives on decaying vegetation and releases
large amounts of conidia, which are dispersed by air currents and which
are present in all aerial environments (10). Patient
infection is acquired upon inhalation of conidia. Identification of
infective strains and contaminating sources has been a major
epidemiological concern in the study of nosocomial aspergillosis due to
A. fumigatus (14).
Three DNA fingerprinting techniques have been extensively used for
typing A. fumigatus isolates (see reference 14 for early references). The most popular technique is based on random amplified polymorphic DNA (RAPD) PCR using short single primers and
low-stringency conditions (1, 2, 15, 18). The second
technique is based on restriction fragment length polymorphism (RFLP)
analysis upon Southern blot hybridization with inactive retrotransposon
Afut1 (20). By using this moderately repetitive
species-specific sequence, extremely high levels of genetic diversity
within A. fumigatus have been revealed (9, 11).
The third technique is based on PCR amplification of
(CA)n repeats isolated from an A. fumigatus genomic library to detect microsatellite length
polymorphism (MLP) (3, 4). In view of the discrepancies
previously reported between RAPD and RFLP methods (1, 17,
26) and the recent development of MLP analysis, a comparison
among these three fingerprinting systems was needed to identify the
most accurate genotyping method currently available for A. fumigatus.
Sixty-seven A. fumigatus clinical and environmental
isolates, including three subcultured reference strains, were randomly selected from two isolate collections (Table
1). With two exceptions, clinical
isolates were recovered from different individuals cared for either in
a single hospital at different time periods or in distinct
institutions. All environmental isolates differed by their location and
time of recovery, and none was related to a patient stay. DNA was
purified at the Unité des Aspergillus (Pasteur Institute, Paris,
France) as previously described (9). Aliquots from single
DNA batches were given an arbitrary code and sent to each of the three
collaborating laboratories. To obtain the most-reliable results, each
laboratory carried out its familiar fingerprinting technique. RFLP
typing (performed at the Institut Pasteur) consisted of electrophoresis
of EcoRI restriction fragments, Southern blot hybridization
with retrotransposon-like sequence Afut1, and computerized
analysis of the banding patterns visualized on the autoradiographs
using GelCompar software (8, 9). A visual examination of
the autoradiographs was systematically performed to validate the
software data (9). Two isolates were considered different
when the hybridization patterns differed by at least two bands. MLP
typing (performed at Hopital Henri Mondor) consisted of PCR
amplification of four A. fumigatus CA-containing microsatellite sequences using specific primer sets and precise sizing
of PCR products with an automatic sequencer (3). A single size difference among the microsatellite loci studied led to a different genotype. RAPD typing was performed as previously described at the Erasmus Medical Center by using enterobacterial intergenic consensus primers ERIC-1 and ERIC-2 and decamers RAPD-1281 and RAPD-1283 in two combination assays (15, 16). The patterns were compared visually; differences in ethidium bromide staining intensities were ignored, and a single band difference led to a
different overall genotype (15). For each technique, data were obtained from two experiments. Four independent investigators compared the three series of data before scoring for isolate identity.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2683-2686.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Comparison of Restriction Fragment Length
Polymorphism, Microsatellite Length Polymorphism, and Random
Amplification of Polymorphic DNA Analyses for Fingerprinting
Aspergillus fumigatus Isolates
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TABLE 1.
Genotypes obtained by RFLP, MLP, and RAPD for the 67 A. fumigatus isolates studied
Typing results are shown in Table 1. Among the 67 isolates studied,
RFLP analysis generated 49 genotypes, of which 37 were unique. MLP
analysis yielded 43 genotypes including 28 unique types. RAPD analysis
detected 31 distinct genotypes, with 17 unique types. Both RFLP and MLP
methods generated an identical discrimination for 53 isolates and 33 genotypes (Tables 1 and 2). Statistical analysis, based on the comparison of nonunique genotypes (Table 2),
indicated a significant concordance between RFLP and MLP (coefficient
of contingency [C] = 0.91; P < 0.001).
Among the 14 discrepancies observed, 11 corresponded to isolates
displaying unique profiles only by RFLP; 8 of these unique types were
assigned on the basis of differences in two discrete bands. The other
three discrepancies (isolates IP32, IP33, and IP27) resulted from a higher differentiation by MLP due to one marker. Levels of
concordance between RAPD and either RFLP or MLP were similarly low:
perfectly concordant genotypes were only found for 13 and 12 genotypes
respectively (corresponding to only 15 and 14 isolates, respectively)
(Tables 1 and 2). In addition, two (or more) genotypes defined by RFLP or by MLP could not be related to one RAPD genotype. For
instance, RAPD genotypes I to VI included isolates displaying both
unique and nonunique RFLP patterns (Table 1). As a result, no
significant concordance was obtained, either between RFLP and RAPD
(C = 0.84; P > 0.05) or between MLP
and RAPD (C = 0.83; P > 0.05).
Additionally, six and seven isolates were found unique by RAPD but not
by RFLP and MLP, respectively (isolates IP37, IP39, IP48, IP49, IP33, Cr60, and Cr55). In all cases, the uniqueness of the RAPD types was due
to differences in two or more RAPD bands.
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This study is the first that compares the three completely different DNA fingerprinting methods with the highest discriminatory levels so far reported for A. fumigatus (3, 8, 16). The wide genetic diversity of this fungal species is reflected not only in strains from distinct sources and geographical locations but also within isolate populations sampled in one hospital, as 85% of the environmental isolates may be recovered only once (8). In the present study of 67 isolates originating from various environmental and clinical sources, each fingerprinting system was expected to detect a consistent level of genetic polymorphism. This polymorphism was best detected with RFLP and with MLP, not with the RAPD system, which generated the lowest variety of fingerprints. Concordance among these fingerprinting systems was also expected, and MLP and RFLP provided identical differentiation for 78% of the isolates and concordant types for 67%. RAPD had a considerably lower discriminatory ability with identical differentiation for 21 and 22% of the isolates when compared, respectively, with RFLP and MLP and did not generate data significantly concordant with that generated with MLP or with RFLP. This was either because RAPD did not differentiate isolates defined as unique by RFLP or by MLP or because it differentiated isolates considered nonunique by the other two systems. Thus, our results suggest that, in most cases, isolate discrimination by RAPD fingerprinting will be erroneous.
Discordant results between RAPD and RFLP hybridization have indeed been previously reported (1, 26), and many factors can be implicated in the explanation. First, PCR components and parameters and DNA quality and gel electrophoresis duration are known to affect the reproducibility of RAPD, as does the discriminatory strength of selected primers (17, 19, 24). In our study, DNA quality does not seem to interfere with the RAPD patterns since no modification of the patterns was seen when a DNA extraction protocol currently used for RAPD was tested on 10 isolates (26). Second, visual comparison of the banding patterns of different gels may be subjective, even in the presence of internal standards (25). Third, a band may consist of different RAPD products with equal electrophoretic mobilities (21). Fourth, the appearance or disappearance of a RAPD band is more likely to result from nonspecific primer hybridization to the DNA template during amplification than from some mutation event at a specific priming site (5, 28).
In contrast to RAPD fingerprinting, the MLP and RFLP genotyping systems are based on specific DNA hybridization between either a probe or primer(s) and the target DNA. The reproducibility of these techniques is therefore high. Moreover, both methods detect definite phenomena: either mutations in enzyme restriction sites for the RFLP system or differences in the number of CA repeats for the MLP system (3). Under the assumption of a repartition of these phenomena in the A. fumigatus genome, the congruence between the two techniques is not surprising. Presently, RFLP remains the more discriminating system, although complex RFLP patterns can be difficult to interpret (9). In the future, the discriminatory power of the MLP system will be probably improved by adding polymorphic microsatellite sequences to be identified in the ongoing sequencing project of the A. fumigatus genome, as seen recently for Saccharomyces cerevisiae (13). MLP analysis is easy since a single PCR band product is obtained, in accordance with the haploidy of the A. fumigatus genome. An additional advantage of the MLP typing system over the RFLP and RAPD methods is its capacity to detect a mixture of isolates (3). Indeed, two original isolates were rejected from our study upon detection of distinct PCR band products at a single locus and confirmation of their cross-contamination upon monospore subculturing. However, some artifacts may occur during amplification of dinucleotide repeats and must be overcome by optimization of PCR conditions (7, 12) and monitored by including well-characterized strains in each typing (3). MLP and RFLP have been also successfully used to type other human pathogens (6, 23).
Regarding our results, the very convenient RAPD approach should be not favored for A. fumigatus, even as a first-line typing strategy. In contrast, because of the methodology used, MLP analysis can give quick, reliable, computerizable data and may be considered the first-line choice for typing A. fumigatus isolates, despite its high cost. If more discrimination is needed, RFLP, presently the most powerful technique of the three tested in this study, should be used.
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ACKNOWLEDGMENTS |
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We are grateful to J. Cabaret from the Station de Pathologie Aviaire et de Parasitologie (INRA; Nouzilly, France) for assisting with the statistical analysis.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité des Aspergillus, Institut Pasteur, 25, rue du docteur Roux, 75724 Paris cedex, France. Phone: 33 1 40 61 35 18. Fax: 33 1 40 61 34 19. E-mail: jplatge{at}pasteur.fr.
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