Medical Mycology October 2004, 42, 433 /437

PCR identication system for the genus Aspergillus and three major pathogenic species: Aspergillus fumigatus, Aspergillus avus and Aspergillus niger
CHISE SUGITA*,$, KOICHI MAKIMURA$, KATSUHISA UCHIDA$, HIDEYO YAMAGUCHI$ & ATSUSHI NAGAI* *First Department of Internal Medicine, Tokyo Womans Medical University, Shinjuku, Tokyo and $Teikyo University Institute of Medical Mycology, Hachioji, Tokyo, Japan

Med Mycol Downloaded from informahealthcare.com by HINARI on 05/07/13 For personal use only.

A PCR system was developed that allowed recognition of three major pathogenic Aspergillus species, namely A . fumigatus , A. niger and A. flavus , in isolates obtained from clinical specimens. The primer pair for PCR was designed from conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA and its flanking regions. Products 521 bp in size were successfully amplified by PCR from the seven Aspergillus species most frequently encountered as opportunistic pathogens, and the three most commonly significant species were identified by separate PCR reactions or nested PCR based on use of species-specific primers. To our knowledge, this is first report of identification of the second and third most frequent pathogenic Aspergillus species using specific PCR amplification. The PCR based identification system reported here will be a powerful tool to control difficult pulmonary fungal infections and to speed the application of effective treatment. Keywords Aspergillus species, identication, internal transcribed spacer 1 ribosomal DNA, nested PCR

Introduction
Aspergillus species have recently caused increasing numbers of life-threatening acute invasive infections in immunocompromized patients [1]. The steadily increasing incidence of invasive aspergillosis over the last few decades is ascribable to the increasing number of patients undergoing chemotherapy, bone marrow or solid organ transplantation and intensive corticosteroid therapy [2]. Conventional diagnosis of fungal infection relies on the identification of pathogens by means of morphological characters specific to the genus and species. This is sometimes unsuccessful, however, because of the atypical features of some isolates. Molecular biological identification systems for pathogenic aspergilli have been suggested as a solution to this problem: for

Received 10 January 2003; Accepted 10 November 2003 Correspondence: K. Makimura, Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192-0395, Japan. Tel.: '/ 81 426 78 3256 Fax: '/ 81 426 74 9190 E-mail: makimura@main.teikyo-u.ac.jp

example, a PCR based diagnostic method for detecting the genus Aspergillus using 18S rDNA [3,4] has been designed. Systems have also been described for specific detection of Aspergillus fumigatus with primers based on regions of the 28S rDNA [5] or of the internal transcribed spacer (ITS) 1 and 2 regions of ribosomal DNA (rDNA) [6 /9]. These PCR systems described to date are useful only in identifying the genus Aspergillus as a whole or the single species A. fumigatus . The ITS region contains variable elements that allow for sequence-based identification of Aspergillus species [10]; therefore, the region offers a possible template for design of species-specific primers for identification of the major pathogenic species. Because the number of species of pathogenic fungi known to infect immunocompromized patients is growing [9], it is essential that quick and reliable methods of identification be found for the most common pathogenic species of aspergilli. This means that not just A. fumigatus , but also Aspergillus flavus and A. niger, should be rapidly identified by a successful system.
DOI: 10.1080/13693780310001656786

2004 ISHAM

434

Sugita et al.

In the present study, we attempted to develop a rapid and reliable identification system to identify the genus Aspergillus in general and the three major pathogens named. We developed an identification system based on new primers, derived from the ITS1 region and its flanking regions, for use in PCR and nested PCR.

DNA preparation
Strains were grown on Sabouraud dextrose agar (SDA; peptone 1%, glucose 1%, agar 1%) at 278C for 1 /5 days depending on strain growth rate. DNA was rapidly prepared from strains of fungi by the method previously described by authors of the present study [3,11]. A small amount of fungal material grown on SDA was placed in lysis buffer (200 mmol/l Tris-HCl pH 8.0, 0.5% sodium dodecyl sulfate [SDS], 250 mmol/l NaCl, 25 mmol/l ethylenediamine tetraacetic acid [EDTA]) and, for molds, crushed with a conical grinder. This material was then incubated at 1008C for 15 min and mixed with 150 ml 3.0 mol/l sodium acetate, kept at (/208C for 10 min, then centrifuged at 10 000 g for 5 min. The supernatant was extracted once with phenol-chloroform-isoamyl alcohol (25:24:1), and subsequently extracted once with chloroform. DNA was precipitated with an equal volume of isopropanol at (/208C for 10 min, washed with 0.5 ml 70% ethanol, dried, and suspended in 50 ml ultra pure water (Milli-Q

Materials and methods

Organisms
Fungal strains used in this study are shown in Table 1. Eighteen species were represented by 27 stock cultures. Included were seven Aspergillus species represented by 16 strains, as well as two fresh clinical isolates. Clinical strain 1 was isolated from a tumor of the lung; strain 2 was an isolate from sputum. In neither isolate could the species be identified by its morphology because the isolates had little or no ability to produce conidia.

Med Mycol Downloaded from informahealthcare.com by HINARI on 05/07/13 For personal use only.

Table 1 Specicity of the primer sets tested in this study. Species Strain Primer set codes ASAP Aspergillus fumigatus TIMM3968 2920 0108 TIMM0057 2935 2912 0117 TIMM0113 0114 2915 2930 2932 TIMM2929 TIMM1290 TIMM2910 TIMM0056 TIMM3834 TIMM0002 TIMM0883 TIMM1293 TIMM0882 TIMM0887 TIMM1304 TIMM3177 TIMM3966 TIMM3173 TIMM1768 '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ Fmi '/ '/ '/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ Fla (/ (/ (/ '/ '/ '/ '/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ Nig (/ (/ (/ (/ (/ (/ (/ '/ '/ '/ '/ '/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ (/ ITS '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/

A. flavus

(A. oryzae ) A. niger

A. terreus A. versicolor A. japonicus A. clavatus Alternaria alternata Absidia corymbifera Penicillium chrysogenum P. expansum P. citreonigrum Pseudallescheria boydii Fusarium solani Mucor circinelloides Trichosporon asahii Cryptococcus neoformans Candida albicans

ASAP, genus Aspergillus specific primer-pair; Fla, A. flavus -specific primer set; Fmi, A. fumigatus -specific primer set; ITS, general fungus specific primer-pair; Nig, A. niger- specific primer set; TIMM, Teikyo University Institute of Medical Mycology.

2004 ISHAM, Medical Mycology, 42, 433 /437

PCR identification system for Aspergillus

435

Synthesis A10; Millipore); 1 ml solution was used as the template for PCR [11].

Oligonucleotide design
All oligonucleotides used in this study were designed based on the relevant sequences of the ITS1 and flanking regions deposited in the DDBJ/EMBL/GenBank database (accession nos.: X78537, A. flavus ; M55626, A. fumigatus ; X78538, A. niger ; X78540, A. terreus ). The following two oligonucleotide primers (termed ASAP) were designed for specific detection of the genus Aspergillus , ASAP1:5?-CAGCGAGTACATCACCTTGG-3? and ASAP2:5?-CCATTGTTGAAAGTTTTAACTGATT-3?. These oligonucleotides and the others mentioned below were made by Amersham Bioscience, Japan. The primers were expected to amplify a fragment 521 bp in length including the whole ITS1 region. Also, three oligonucleotide primer sets based on specific regions within the ASAP1 and ASAP2 amplicons were designed for use in identifying A. fumigatus, A. niger and A. flavus. The A. fumigatusspecific primer set (called Fmi) was made up of primers designated ASPU (5?-ACTACCGATTGAATGGCTCG-3?) and Af3r (5?-CATACTTTCAGAACAGCGTTCA-3?). The A. niger set (called Nig) was composed of ASPU and Ni1r (5?-ACGCTTTCAGACAGTGTTCG-3?), while the A. flavus set (Fla) consisted of ASPU and Fl2r (5?-TTCACTAGATCAGACAGAGT3?). Pan-fungal primers amplifying 18S rDNA regions (B2F; 5?-ACTTTCGATGGTAGGATAG-3? and B4R; 5?-TGATCGTCTTCGATCCCCTA-3? [11]) and ITS1 rDNA (18SF1; 5?-AGGTTTCCGTAGGTGAACCT-3? and 58SR1; 5?-TTCGCTGCGTTCTTCATCGA-3? [12]) were used as positive controls.

and PCR was performed in cycles of 948C for 1 min; 608C for 15 s and 728C for 15 s. There were 25 cycles in total. Thermal cycling was terminated by polymerization at 728C for 10 min. The total cycling process required 1.7 h.

Agarose gel electrophoresis

Between 5 and 10 ml PCR product was electrophoresed in 1.2% agarose gel and visualized by ethidium bromide staining and ultraviolet irradiation.

DNA sequence
PCR products were immediately sequenced by a DNA sequencing kit (Applied Biosystems) with primers for 18S rDNA (B2F and B4R, detailed above) and an automatic sequencer (Genetic Analyzer 310; Applied Biosystems) used according to the manufacturers instructions. The sequences were analyzed with Genetyx-Mac10 software (Software Development, Tokyo, Japan) and searched on the DDBJ/EMBL/GenBank nucleotide database using BLAST programs.

Med Mycol Downloaded from informahealthcare.com by HINARI on 05/07/13 For personal use only.

Results
The genus-level specificity of the putative Aspergillus specific primer system, or ASAP, was tested. A product of 0.5 kbp was amplified by PCR from all tested aspergilli, namely A. fumigatus , A. niger, A. flavus (including its non-toxigenic domesticated form A. oryzae ), A. terreus, A. versicolor, A. japonicus and A. clavatus , but not from other fungi, including Candida albicans , Cryptococcus neoformans, Trichosporon asahii, Mucor circinelloides, Fusarium solani, Pseudallescheria boydii, Penicillium chrysogenum, P. citreonigrum (isolate stored in TIMM under synonymous name P. citreoviride ), P. expansum, Absidia corymbifera and Alternaria alternata (Table 1). The specificity of putatively species-specific primersets for A. fumigatus , A. flavus and A. niger (Fmi, Fla and Nig, respectively), was also tested. Each of these primer-sets was successful in amplifying only the target species (Table 1; Fig. 1). Two nearly or completely nonsporulating clinical isolates from respiratory materials, strains 1 and 2, were subjected to the PCR identification. Strain 1 gave a positive amplification with the 18S rDNA-based panfungal primers, but it was negative by ASAP. This means that it was not an Aspergillus species. Subsequent sequencing and mating analysis showed that the strain was Schizophyllum commune (data not shown). Strain 2 was positive by both pan-fungus PCR and ASAP. Nested PCR showed that it was positive only

PCR
The genus specific ASAP PCR mixture contained 10 ml 10 )/reaction buffer, 100 mM each of dATP, dCTP, dGTP and dTTP, 2.5 U Taq polymerase (all Amersham), 30 pmol of each primer, and 2 ml DNA template solution. Ultra-pure water was added to increase the volume to 100 ml. For ASAP PCR reaction, each mixture was heated to 948C for 4 min and PCR was performed with the following program for the thermal cycler (GeneAmp PCR system 2400, Applied Biosystems): 948C for 1 min; 558C for 2 min; and 728C for 90 s; all repeated for 30 cycles. Thermal cycling was terminated by polymerization at 728C for 10 min. For the species specific primer sets Fmi, Nig, and Fla, PCR, 2 ml DNA solution from isolates or 1 ml of the PCR products diluted 1/100 were used as a template. Each mixture was heated to 948C for 4 min
2004 ISHAM, Medical Mycology, 42, 433 /437

436

Sugita et al.

Fig. 1 Specicity of species-specic primer sets in nested PCR. Lanes left to right depict specic primer set Fmi for Aspergillus fumigatus ; Fla for A. avus ; Nig for A. niger ; ITS1, ITS1-specic panfungal primer set used as a positive control.

Fig. 2 Identication of morphologically unidentiable clinical isolates using the nested PCR system. Strain 1 is a lamentous fungus isolated from a resected pulmonary tumour, and Strain 2 is a mould cultured from sputum. Lanes: M, molecular marker; F, panfungal primer pair (positive control); A, primer pair ASAP specic for Aspergillus at genus level; Fmi, A. fumigatus specic primers as in Fig. 1; Fla, A. avus -specic primers; Nig, A. niger specic primers.

with primer set Fla and was negative with Fmi and Nig (Fig. 2); therefore, it was identified as A. flavus . This result was also confirmed by DNA sequencing (data not shown).

Discussion
Most previous publications on PCR-based detection or identification systems for Aspergillus spp. were based

on using 18S or 28S rDNA as target DNA. However, the sequences in these regions are conserved across a wide range of fungi; it is therefore difficult to design truly species-specific primers. As reported previously, the more variable ITS regions have proven more useful for identification of fungal species [13]. To our knowledge, the present study is the first using specific PCR amplification to allow identification not just of A. fumigatus but also A. flavus and A. niger, the second and third most frequently significant Aspergillus species in opportunistic infection, using specific PCR amplification. The importance of these species should not be underestimated. The number of infections they cause is increasing [9,14 /16]. Species identification of moulds using histopathology or conventional morphology is not always successful. For example, a fungus ball can be produced by a number of filamentous fungi, such as S. commune [17] or P. boydii [18], which are able to grow saprotrophically in the human body. If these fungi grow out as nonsporulating or otherwise atypical mycelium, identification is problematical [17]. The infections cased by these fungi are similar to aspergillosis clinically and pathologically; however, these fungi have poor susceptibility to amphotericin B and candins, so different antifungal drugs must be used for their control. Drug susceptibilities, treatment procedures and prognoses vary widely among fungal species; therefore, reliable techniques for species identification are essential for effective management. The genus Aspergillus consists of about 185 species [19]. A. fumigatus, A. niger and A. flavus account for more than 95% of isolates causing aspergillosis [20]. In the diagnosis of aspergillosis, identification of the species involved is extremely important. Contamination caused by environmental fungi poses a serious problem and Aspergillus species, including many nonpathogens, are ubiquitous. To identify an Aspergillus from clinical specimens as a causal agent often requires corroboration based on repeated isolation of the same fungal species. This must be done reliably in order to exclude confusion arising from successive isolations of various contaminating aspergilli. Numerous recent reports have advocated direct sequencing of PCR products for rapid molecular biological identification of fungal strains. The DNA sequence is superably reliable and informative; however, the necessary equipment is expensive and the process takes more than 2 days. The PCR based identification system reported here is a powerful tool that facilitates diagnosis of infection at an early stage, and allows prompt control of difficult pulmonary
2004 ISHAM, Medical Mycology, 42, 433 /437

Med Mycol Downloaded from informahealthcare.com by HINARI on 05/07/13 For personal use only.

PCR identification system for Aspergillus

437

fungal infections, in part by rapid application of effective treatment.

References
1 Kontoyiannis DP, Bodey GP. Invasive aspergillosis in : an update. Eur J Clin Microbiol Infect Dis 2002; 21: 161 /172. 2 Yamazaki T, Kume H, Murase S, Yamashita E, Arisawa M. Epidemiology of visceral mycoses: analysis of data in annual of the pathological autopsy cases in Japan. J Clin Microbiol 1999; 37: 1732 /1738. 3 Makimura K, Murayama SY, Yamaguchi H. Specic detection of Aspergillus and Penicillium species from respiratory specimens by polymerase chain reaction (PCR). Jpn J Med Sci Biol 1994; 47: 141 /156. 4 Yamakami Y, Hashimoto A, Tokimatsu I, Nasu M. PCR Detection of DNA specic for Aspergillus species in serum of patients with invasive aspergillosis. J Clin Microbiol 1996; 34: 2464 /2468. 5 Spreadbury C, Holden D, Aufauvre-Brown A, Bainbridge B, Cohen J. Detection of Aspergillus fumigatus by polymerase chain reaction. J Clin Microbiol 1993; 31: 615 /621. 6 Gaskell GJ, Carter DA, Britton WJ, Tovey ER, Benyon FH, Lovborg U. Analysis of the internal transcribed spacer regions of ribosomal DNA in common airborne allergenic fungi. Electrophoresis 1997; 18: 1567 /1569. 7 Radford SA, Johnson EM, Leeming JP, et al . Molecular epidemiological study of Aspergillus fumigatus in a bone marrow transplantation unit by PCR amplication of ribosomal intergenic spacer sequences. J Clin Microbiol 1998; 36: 1294 /1299. 8 Zhao J, Kong F, Li R, Wang X, Wa Z, Wang D. Identication of Aspergillus fumigatus and related species by nested PCR targeting ribosomal DNA internal transcribed spacer regions. J Clin Microbiol 2001; 9: 2261 /2266. 9 Henry T, Iwen PC, Hinrichs SH. Identication of Aspergillus species using internal transcribed spacer regions 1 and 2. J Clin Microbiol 2000; 38: 1510 /1515.

Med Mycol Downloaded from informahealthcare.com by HINARI on 05/07/13 For personal use only.

10 Iwen PC, Hinrichs SH, Rupp ME. Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens. Med Mycol 2002; 40: 87 /109. 11 Makimura K, Murayama SY, Yamaguchi H. Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 1994; 40: 358 /364. 12 Makimura K, Tamura Y, Mochizuki M, et al . Phylogenetic classication and species identication of dermatophyte strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol 1999; 37: 920 /924. 13 Makimura K. Species identication system for dermatophytes based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1. Nippon Ishinkin Gakkai Zasshi 2001; 42: 61 /67. 14 Hoshino H, Tagaki S, Kon H, et al . Allergic bronchopulmonary aspergillosis due to Aspergillus niger without bronchial asthma. Respiration 1999; 66: 369 /372. 15 Nenoff P, Kliem C, Mittag M, Horn LC, Niederwieser D, Haustein UF. Secondary cutaneous aspergillosis due to Aspergillus avus in an acute myeloid leukaemia patient following stem cell transplantation. Eur J Dermatol 2002; 12: 93 /98. 16 Rao K, Saha V. Medical management of Aspergillus avus endocarditis. Pediatr Hematol Oncol 2000; 17: 425 /427. 17 Sigler L, de la Maza LM, Tan G, Egger KN, Sherburne RK. Diagnostic difculties caused by a non-clamped Schizophyllum commune isolate in a case of fungus ball of the lung. J Clin Microbiol 1995; 33: 1979 /1983. 18 Khurshid A, Theodore V, Sekosan M, Ginzburg AS, Onal E. Disseminated Pseudallescheria boydii infection in a non-immunocompromised host. Chest 1999; 116: 572 /574. 19 Kirk PM, Cannon PF, David JC. 2001 Stapler Dictionary of the Fungi , 9th edn. Egham, UK: CABI Bioscience. 20 Richardson MD, Ajello L, Hay RJ. Volume 4 of Collier L, Balows A, Sussman M, eds. Topley and Wilsons Microbiology and Microbial Infections , 9th edn. New York: Arnold, 1995; 281 /312.

2004 ISHAM, Medical Mycology, 42, 433 /437