Comparison of Restriction Fragment Length Polymorphism

JOURNAL OF CLINICAL MICROBIOLOGY, July 2001, p. 2683–2686 0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.7.2683–2686.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 39, No. 7

Comparison of Restriction Fragment Length Polymorphism, Microsatellite Length Polymorphism, and Random Amplification of Polymorphic DNA Analyses for Fingerprinting Aspergillus fumigatus Isolates EMMANUELLE BART-DELABESSE,1 JACQUELINE SARFATI,2 JEAN-PAUL DEBEAUPUIS,2 WILLEM VAN LEEUWEN,3 ALEX VAN BELKUM,3 STEPHANE BRETAGNE,1 2 AND JEAN-PAUL LATGE * Laboratoire de Parasitologie-Mycologie, Ho ˆpital Henri Mondor, Cre´teil,1 and Unite´ des Aspergillus, Institut Pasteur, 2 Paris, 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 indi-

viduals 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 Unite´ 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 RAPD1283 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. 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,

* Corresponding author. Mailing address: Unite´ 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@pasteur .fr. 2683

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TABLE 1. Genotypes obtained by RFLP, MLP, and RAPD for the 67 A. fumigatus isolates studied Isolate

IP5 IP6 IP8 IP7 IP24 IP23 IP30 IP31 IP33 IP38 IP35 IP36 IP37 IP39 IP27 IP32 IP42 IP43 IP48 IP49 IP18 IP17 IP41 IP50 Cr53 Cr52 Cr60 Cr59 Cr66 Cr67 Cr55 IP26 IP40 Cr71 IP3 IP2 Cr51 IP45 IP46 Cr61 IP16 IP1 IP14 IP47 Cr54 Cr73 Cr74 IP4 IP21 IP11 IP12 IP13 Cr70 Cr72 Cr62 Cr63 IP15 Cr65 IP10 IP22 IP25 Cr56 Cr57 Cr58 Cr64 Cr68 Cr69

Origin

Type by:

a

Env (Hosp.1) Env (Hosp.1) Pat (Hosp.2) Pat (Hosp.2) Env (Hosp.2) Env (Hosp.1) Env (Hosp.1) Env (Hosp.1) Env (Hosp.1) Pat (The Netherlands) Env (Hosp.1) Env (Hosp.1) Pat (Hosp.2) Pat (Germany) Env (Hosp.2) Env (Hosp.1) Env (Hosp.1) Env (Hosp.2) Env (Hosp.1) Env (Hosp.2) Env (Hosp.1) Env (Hosp.2) Bovineb Bovineb Env (Hosp.3) Env (Hosp.3) Pat (Hosp.3) Pat (Hosp.3) Pat (Hosp.3)b Pat (Hosp.3)b Env (Hosp.3) Pat (Germany) Pat (Hosp.2) Pat (Hosp.3) Pat (Hosp.2) Pat (CBS 144-89) Pat (Hosp.3) Env (Hosp.1) Env (Hosp.1) Env (Hosp.3) Env (Hosp.1) Pat (Hosp.2) Env (Hosp.1) Pat (Germany) Pat (Hosp.3) Pat (Hosp.3) Env (Hosp.3) Env (Hosp.1) Env (Hosp.2) Env (Hosp.1) Env (Hosp.1) Env (Hosp.1) Pat (Hosp.3) Pat (IP 2279-94) Pat (Hosp.3) Env (Hosp.3) Env (Hosp.1) Env (Hosp.3) Pat (Hosp.3) Bovine Pat (CBS 143-89) Env (Hosp.3) Pat (Hosp.3) Env (Hosp.3) Env (Hosp.3) Pat (Hosp.3) Pat (Hosp.3)

RFLP

MLP

RAPD

A A B B C C D D D E E E E E E E F F G G H H I I J J K K L L Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique

a a b b c c d d Unique e e e e e f Unique g g h h i i j j m m n n o o b f j j k k n l l n n Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique Unique

I I I II III IV V V Unique I II II Unique Unique VI II V V Unique Unique IV XIII VII VII II X Unique XIII XI XI Unique VI Unique III I II I VIII VIII XII IV II II II II II II III IV V IX IX X X XII XII XIV XIV Unique Unique Unique Unique Unique Unique Unique Unique Unique

a Hosp.1, Ho ˆpital Ho ˆtel-Dieu, Paris, France; Hosp.2, Ho ˆpital Trousseau, Paris, France; Hosp.3, Ho ˆpital Henri-Mondor, Cre´teil, France; env, environmental; pat, patient; IP, Institut Pasteur Collection; Cr, Creteil Collection; CBS, Centraalbureau voor Schimmelcultures. b Isolates IP41 and IP 50 are from the same animal; isolates Cr66 and Cr67 originated from the same patient.

TABLE 2. Concordance between RFLP, MLP, and RAPD upon analysis of 67 A. fumigatus isolates No. of concordant genotypes Methods compared

Total (%)a

Unique

Nonuniqueb

MLP and RFLP RAPD and RFLP RAPD and MLP

33 (67.5) 13 (26.5) 12 (27.9)

26 11 10

7 2 2

Cc (P)

0.91 (⬍0.001) 0.84 (⬎0.05) 0.83 (⬎0.05)

a Percentage calculated according to the method generating the highest number of genotypes. b Each of these genotypes included two isolates. c C was calculated from ␹2 established on a contingency table with k columns and r lines, and the Yates correction was used (21). The significance of C was estimated from the ␹2 values using (k ⫺ 1)(r ⫺ 1) degrees of freedom.

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. 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

VOL. 39, 2001

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,

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