Assessment of Ribosomal Large-Subunit

D1-D2, Internal Transcribed Spacer 1, and

Internal Transcribed Spacer 2 Regions as
Targets for Molecular Identification of
Medically Important Aspergillus Species
Hans P. Hinrikson, Steven F. Hurst, Timothy J. Lott, David
W. Warnock and Christine J. Morrison
J. Clin. Microbiol. 2005, 43(5):2092. DOI:

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10.1128/JCM.43.5.2092-2103.2005.

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JOURNAL OF CLINICAL MICROBIOLOGY, May 2005, p. 2092–2103 Vol. 43, No. 5
0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.5.2092–2103.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Assessment of Ribosomal Large-Subunit D1-D2, Internal Transcribed

Spacer 1, and Internal Transcribed Spacer 2 Regions as Targets for
Molecular Identification of Medically Important Aspergillus Species
Hans P. Hinrikson,† Steven F. Hurst, Timothy J. Lott, David W. Warnock,
and Christine J. Morrison*
Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia

Downloaded from http://jcm.asm.org/ on November 5, 2012 by UNIV OF SUSSEX

Received 25 October 2004/Accepted 27 December 2004

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi
compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2
region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for
the molecular identification of some fungi. We therefore conducted an assessment of these regions for the
identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) cheva-
lieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus
(Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus
ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate
among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated
91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6
to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13
species examined exhibited a <1-nucleotide divergence in the D1-D2 region from closely related but different
species. In contrast, only 5 of the species examined exhibited a <1-nucleotide divergence from sibling species
in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for
some species, and major improvement in the quality and accuracy of GenBank entries is needed, current
identification of medically important Aspergillus species using GenBank reference data seems more reliable
using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.

Aspergillus species are an increasingly important cause of accomplish, and in a small number of cases, isolates may not
invasive fungal infections in immunocompromised patients conidiate, obstructing species identification (20, 38). There-
(31, 59). Unfortunately, there are few specific clinical signs of fore, rapid molecular approaches are now being developed to
invasive aspergillosis and current methods for laboratory diag- replace the need for culture by detecting and identifying As-
nosis are less than ideal, particularly in the early stages of the pergillus species DNA directly from clinical materials.
disease (8, 49). Given the recent reports of reduced antifungal A number of targets for the molecular identification of as-
drug susceptibilities among some Aspergillus species (21, 26, pergilli have been investigated including the mitochondrial cy-
50), the timely and accurate identification of aspergilli to the tochrome b gene (46, 57, 62), a putative aflatoxin pathway
species level has become especially important (10). Species regulatory gene (aflR) (4), the DNA topoisomerase II gene
identification is also important for epidemiological purposes (TOP2) (19), the ␤-tubulin gene (11), and various rRNA gene
and as a guide to clinical management (29, 47, 48). regions (18). The most promising targets to date have been the
The current laboratory identification of Aspergillus species is 5⬘ end of the large-subunit rRNA gene (D1-D2 region) (35)
based on macroscopic colonial and microscopic morphological and the internal transcribed spacer 1 and 2 (ITS1 and ITS2)
characteristics (7, 20, 45). Over 180 different species in at least regions between the small- and large-subunit rRNA genes (18,
16 subgeneric groups or sections can be distinguished (35, 37, 60). The use of DNA sequence diversity in the ribosomal re-
38), including approximately 30 species which are recognized gions as an aid to species identification has been exploited
as opportunistic pathogens of humans (7). Many clinical lab- using PCR amplification of targets followed by either fragment
oratories use traditional phenotypic methods of identification length analysis (1, 16, 22, 39, 52, 56), DNA probe hybridization
and can differentiate only the more common Aspergillus spe- (15, 30, 32, 61), or DNA sequence analysis (3, 17, 35, 43, 55,
cies; the delineation of less common species must be referred 61). In general, DNA-based approaches were found to provide
to specialist laboratories. In addition, species identification by more reliable and faster species identifications than culture-
traditional phenotypic methods may require several weeks to
based methods. Nonetheless, some diagnostic problems were
encountered, resulting from similar amplicon or fragment
* Corresponding author. Mailing address: Mycotic Diseases Branch, lengths for different taxa (16, 39, 52), unexpected cross-hybrid-
Centers for Disease Control and Prevention, 1600 Clifton Road, NE, ization results (9, 16, 30, 32, 42, 61), and ambiguous sequenc-
Mail Stop G-11, Atlanta, Georgia 30333. Phone: (404) 639-3098. Fax:
(404) 639-3546. E-mail: [email protected].
ing-based identifications because of significant intraspecies
† Present address: Institut de Microbiologie, Centre Hospitalier heterogeneity (44, 53, 54) or virtually identical sequences for
Universitaire Vaudois, 1011 Lausanne, Switzerland. apparently distinct organisms (14, 17, 35, 43). The validation of

2092
VOL. 43, 2005 RIBOSOMAL IDENTIFICATION OF ASPERGILLUS SPECIES 2093

test specificity was further compromised by the absence of a primer pair D1 and D2R for the seminested PCR amplification included dena-
comprehensive test strain panel; most studies did not include turation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 60°C for 45 s,
and 72°C for 90 s. A final extension step at 72°C for 7 min was then employed.
type strains or authenticated culture collection reference PCR amplification of the ribosomal ITS1 and ITS2 regions. The broad-range
strains of the target species or of closely related species in their primer pairs ITS5 (5⬘ GGA AGT AAA AGT CGT AAC AAG G) and ITS2 (5⬘
examinations. Finally, although the D1-D2 and the ITS1 and GCT GCG TTC TTC ATC GAT GC) or ITS3 (5⬘ GCA TCG ATG AAG AAC
ITS2 regions have been analyzed separately for some species of GCA GC) and ITS4 (5⬘ TCC TCC GCT TAT TGA TAT GC) were used to
amplify ribosomal internal transcribed spacer regions 1 and 2, respectively (60).
Aspergillus, a systematic evaluation and comparison of se-
Some samples required seminested PCR amplification with primer pair ITS5 and
quences from all three ribosomal regions for their usefulness in ITS4 followed by ITS5 and ITS2 or ITS3 and ITS4 to generate sufficient quan-
the identification and differentiation of the most medically tities of PCR amplicon for DNA sequencing. Amplifications and reamplifications
important Aspergillus species has not been published to date. were performed as described above, except for the primer pair used and minor
Therefore, the present investigation assessed the utility of differences in the thermal cycling conditions employed: initial denaturation at
95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for
the ribosomal D1-D2, ITS1, and ITS2 regions as targets for the

Downloaded from http://jcm.asm.org/ on November 5, 2012 by UNIV OF SUSSEX

1 min. A final extension step at 72°C for 5 min was then conducted.
molecular identification of 13 potentially invasive Aspergillus DNA sequencing. All PCR products were purified before DNA sequence
species (Aspergillus candidus, Aspergillus chevalieri, Aspergillus analysis using a QIAquick PCR purification kit (Qiagen) according to the man-
flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus ufacturer’s instructions. Purified amplicons were then sequenced on both strands
using the same primers as described above. BigDye terminator cycle sequencing
granulosus, Aspergillus nidulans, Aspergillus niger, Aspergillus
Ready Reaction kits (Perkin Elmer Applied Biosystems) were employed as
restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus us- recommended by the manufacturer. All cycle sequencing reactions were per-
tus, and Aspergillus versicolor). We acquired DNA sequence formed on a GeneAmp model 9700 thermal cycler using an initial denaturation
information concerning all three ribosomal regions for each of at 96°C for 5 s, followed by 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for
the above species and used this information to conduct DNA 4 min. Products were purified using a Dye-Ex spin kit (Qiagen), dried in a
vacuum centrifuge, and resuspended in either template suppression reagent
sequence alignments, pairwise nucleotide sequence analyses, (Perkin Elmer Applied Biosystems; D1/D2R products) or formamide (Perkin
comparative GenBank database searches, and an examination Elmer Applied Biosystems; ITS products). Products were then analyzed on an
for sequence length polymorphisms among Aspergillus species. automated capillary DNA sequencer (ABI Prism 310 genetic analyzer; Perkin
Elmer Applied Biosystems) according to the manufacturer’s directions. Com-
parative sequence analysis and GenBank searches were assisted by the Genetics
MATERIALS AND METHODS Computer Group software package (FASTA, BESTFIT, STRETCHER, and
Microorganisms. A total of 36 reference strains, including both type strains PILEUP algorithms; University of Wisconsin, Madison), the Clustal W align-
and authenticated culture collection strains for the following medically important ment program (51), and the nucleotide-nucleotide Basic Local Alignment Search
Aspergillus species, were investigated: A. candidus, A. (Eurotium) chevalieri, A. Tool (BLAST) algorithm (blastn) (2, 63). Calculation of pairwise sequence
(Fennellia) flavipes, A. flavus, A. fumigatus, A. granulosus, A. (Emericella) nidu- identity was restricted to full-length reference data of Aspergillus species cur-
lans, A. niger, A. restrictus, A. sydowii, A. terreus, A. ustus, and A. versicolor rently sanctioned by the International Commission of Penicillium and Aspergillus
(Table 1). Isolates were grown on Czapek-Dox agar (Difco Laboratories, De- (37). Authoritative Aspergillus reference sequences were taken from the work of
troit, Mich.) for up to 14 days at 25°C and up to 1 week at 37°C to confirm their Peterson (35), Haugland et al. (15), and Pazoutova et al. (34).
purity and species identity based on traditional criteria (38), and to generate Nucleotide sequence accession numbers. The Aspergillus large-subunit D1-D2,
fungal growth for DNA extraction. ITS1, and ITS2 ribosomal regions determined in this study were deposited in
DNA extraction. Fungal biomass, scraped from the surface of one agar plate, GenBank and assigned the accession numbers listed in Table 1. The species
was transferred into a precooled (⫺20°C) sterile ceramic mortar, overlaid with designations are in compliance with currently sanctioned names published by the
liquid nitrogen, and carefully ground with a sterile ceramic pestle into a fine International Commission of Penicillium and Aspergillus (37).
powder. The powder was suspended in 2 ml of buffer G-2 (genomic DNA buffer
set; Qiagen, Valencia, Calif.) containing RNase (200 ␮g/ml; Sigma Chemical
RESULTS
Company, St. Louis, Mo.) and transferred into a clean test tube. Following
addition of 45 ␮l of proteinase K solution (20-mg/ml stock solution; Sigma), the Traditional and molecular classification of the Aspergillus
suspension was incubated with intermittent agitation at 55°C for 3 h. The crude
extract was centrifuged at 21,500 ⫻ g for 10 min, and the supernatant was
strains used in this study. Type strains and authenticated
transferred into a clean test tube. DNA was then purified using Genomic-tip culture collection reference strains, representing 13 of the
20/G columns (Qiagen) according to the manufacturer’s instructions. The eluted most common and clinically relevant Aspergillus species, were
DNA was supplemented with 2.5 ␮l of glycogen solution (20 mg/ml; Gentra used for DNA sequence analysis of the D1-D2, ITS1, and ITS2
Systems, Minneapolis, Minn.), precipitated by standard methods using isopro-
ribosomal regions. Species designations were based on culture
panol and ethanol (41), and resuspended in 60 ␮l of DNA rehydration buffer
(PureGene kit, Gentra Systems). DNA was then stored at ⫺20°C until used. collection information provided with the isolates and on phe-
PCR amplification of the D1-D2 region of the large-subunit (28S) rRNA gene. notypic reconfirmation according to Raper and Fennell (38) at
Seminested PCR using broad-range primer pairs ITS1 (5⬘ TCC GTA GGT GAA the Centers for Disease Control and Prevention, Atlanta,
CCT GCG G) and D2R (5⬘ TTG GTC CGT GTT TCA AGA CG) followed by Georgia. Strain designations and the corresponding GenBank
D1 (5⬘ GCA TAT CAA TAA GCG GAG GA) and D2R generated the D1-D2
region amplicons for sequencing (36, 60). PCR amplification was conducted in a
accession numbers for our DNA sequences are listed in Table
GeneAmp model 9700 thermal cycler (Perkin Elmer Applied Biosystems, Foster 1. Strains marked with a superscript “T” represent type strains
City, Calif.), and PCR components were obtained from Roche Molecular Bio- according to information provided on the website of the Ag-
chemicals, Indianapolis, Ind. The PCR mix contained 0.2 mM concentrations of ricultural Research Service Culture Collection (http://nrrl
each deoxynucleoside triphosphate, 1.25 U of Taq DNA polymerase, 0.2 ␮M
.ncaur.usda.gov); strains marked with a superscript “MT” rep-
concentrations of each primer, 2 ␮l of a 1:50 dilution of the original DNA extract
(⬎5 ng of DNA for the initial PCR or 2 ␮l of undiluted amplicon from the initial resent non-type strains with sequences that perfectly matched
PCR when products were reamplified in the seminested PCR), and the appro- the type strain reference data in GenBank (15, 35) in all ribo-
priate amount of PCR buffer (final concentration, 10 mM Tris-HCl [pH 8.3], 1.5 somal regions investigated. For the purposes of this study,
mM MgCl2, 50 mM KCl) to bring the final volume to 50 ␮l. One sample without strains which exhibited minor sequence divergence compared
DNA template was always included as a negative control. Amplification with the
primer pair ITS1 and D2R was performed using a denaturation step of 95°C for
to the corresponding type strain sequences, as determined in
5 min, followed by 30 cycles of 95°C for 30 s, 51°C for 30 s, and 72°C for 150 s. this study, or compared to the corresponding type strain ref-
A final extension step at 72°C for 10 min was then used. Reamplification with erence sequences available in GenBank (15, 35) are designated
2094 HINRIKSON ET AL. J. CLIN. MICROBIOL.

TABLE 1. Ribosomal Aspergillus sequences determined in this study

GenBank accession no. (length in nucleotides)b per target Total size of
Species and sequevara Strainc
D1-D2 ITS1 ITS2 ITS regiond

A. candidus NRRL 303T AF454148 AF453881 (180) AF454098 (168) 505

NRRL 312MT AF454149 AF453882 (180) AF454099 (168) 505

A. (Eurotium) chevalieri ATCC 16443T AF454150 AF453883 (142) AF454100 (167) 466
ATCC 24546MT AF454151 AF453884 (142) AF454101 (167) 466

A. (Fennellia) flavipes I ATCC 11013 AF454154e AF453886 (185)e AF454104 (163)e 505
A. (Fennellia) flavipes II ATCC 16805 AF454155e AF453887 (184)e AF454105 (160)e 501
A. (Fennellia) flavipes III ATCC 24487T AF454156 AF453888 (185) AF454106 (162) 504

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A. flavus ATCC 11497MT AF454158 AF453890 (181) AF454108 (169) 507
ATCC 34896MT AF454159 AF453891 (181) AF454109 (169) 507
ATCC 44310MT AF454160 AF453892 (181) AF454110 (169) 507
ATCC 64025MT AF454161 AF453893 (181) AF454111 (169) 507

A. fumigatus ATCC 1022T AY660917 AY660920 (184) AY660923 (168) 509

ATCC 16903MT AF454163 AF453895 (184) AF454113 (168) 509
CDC B2570MT AF454164 AF453896 (184) AF454114 (168) 509

A. granulosus CBS 119.58MT AF454165 AF453897 (156) AF454115 (171) 484

NRRL 1932T AF454166 AF453898 (156) AF454116 (171) 484

A. (Emericella) nidulans I ATCC 10074T AY660918 AY660921 (153) AY660924 (168) 478
A. (Emericella) nidulans II ATCC 16855 AF454167 AF453899 (153)e AF454117 (168) 478
A. (Emericella) nidulans III CDC B6597 AF454192e AF453924 (154)e AF454141 (169)e 480

A. niger I ATCC 1015MT AF454169 AF453901 (185) AF454118 (169) 511

ATCC 64028MT AF454171 AF453903 (185) AF454120 (169) 511
A. niger II ATCC 16404 AF454170 AF453902 (185)e AF454119 (170)e 512

A. restrictus NRRL 148MT AF454175 AF453907 (181) AF454124 (172) 510

NRRL 151MT AF454176 AF453908 (181) AF454125 (172) 510

A. sydowii I NRRL 250 AF454177e AF453909 (155)e AF454126 (168)e 480

NRRL 4768 AF454178e AF453910 (155)e AF454127 (168)e 480
A. sydowii II NRRL 254T AY660919 AY660922 (155) AY660925 (168) 480

A. terreus ATCC 1012T AF454183 AF453915 (186) AF454132 (177) 520

ATCC 1002MT AF454184 AF453916 (186) AF454133 (177) 520
ATCC 7860MT AF454185 AF453917 (186) AF454134 (177) 520

A. ustus I ATCC 14417 AF454186e AF453918 (155)e AF454135 (170)e 482

A. ustus II ATCC 16801 AF454187e AF453919 (155)e AF454136 (170)e 482
A. ustus III NRRL 275T AF454188 AF453920 (161) AF454137 (172) 490

A. versicolor ATCC 10072MT AF454193 AF453925 (155) AF454142 (167) 479

NRRL 238T AF454194 AF453926 (155) AF454143 (167) 479
NRRL 239MT AF454195 AF453927 (155) AF454144 (167) 479
a
Roman numerals placed after a species name represent different ribosomal sequevars of the same species (minor divergence in one or more ribosomal regions
compared to the corresponding type strain sequences, as determined in this study, or compared to the corresponding type strain reference sequences available in
GenBank; see Results).
b
Sequences containing the D1-D2 region were 542 nt in length for all species except A. nidulans (543 nt).
c
A superscript “T” designates type strains according to information provided on the website of the Agricultural Research Service Culture Collection (http:
//nrrl.ncaur.usda.gov); “MT” designates non-type strains that gave sequence information that perfectly matched the type strain reference data (15, 35) in all ribosomal
regions investigated in this study. ATCC, American Type Culture Collection, Manassas, Va; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands;
CDC, Centers for Disease Control and Prevention, Atlanta, Ga.; NRRL, National Regional Reference Laboratory, Peoria, III.
d
Length of complete ITS1-ITS2 region (including the intervening 5.8S rRNA gene that exhibited a conserved length of 157 nt in all species investigated in this study).
Boldface type indicates overall sequence length for type strain sequences or sequences matching type strain reference data (15).
e
Sequences in these target regions did not match those for the corresponding type strain.

in Table 1 by roman numerals after the species name to rep- study. The D1-D2 sequencing results for all Aspergillus species
resent different sequevars of the same species; ribosomal re- studied showed identical overall lengths for all species and
gions where divergence from the type strain sequences oc- strains investigated (i.e., 542 nucleotides [nt]) with the excep-
curred were identified in Table 1 with a superscript “e.” tion of A. nidulans (total length, 543 nt [Table 1]). In contrast,
Use of the D1-D2, ITS1, and ITS2 DNA sequence length to the ITS1 region ranged in overall length from 142 nt (A. cheva-
discriminate among Aspergillus species investigated in this lieri) to 186 nt (A. terreus), and sequence length was more
VOL. 43, 2005 RIBOSOMAL IDENTIFICATION OF ASPERGILLUS SPECIES 2095

TABLE 2. Pairwise sequence comparison in D1-D2 regions between medically important Aspergillus species investigated in this study

Species % Sequence identity with species no.a:

Species and strainb
no. 1 2 3 4 5 6 7 8 9 10 11 12 13

1 A. fumigatus ATCC 1022 100

2 A. flavus ATCC 11497 94.3 100
3 A. niger ATCC 1015 94.6 96.7 100
4 A. terreus ATCC 1012 95.6 95.2 95.8 100
5 A. flavipes ATCC 24487 95.9 96.5 95.8 96.5 100
6 A. candidus NRRL 303 95.6 96.9 96.1 95.6 96.7 100
7 A. restrictus NRRL 148 93.4 92.8 93.4 93.5 93.4 93.2 100
8 A. chevalieri ATCC 16443 94.5 94.5 94.5 94.6 95.0 94.6 95.8 100
9 A. granulosus NRRL 1932 93.9 94.5 95.2 93.4 95.0 93.9 93.0 94.3 100

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10 A. ustus NRRL 275 93.5 94.1 94.8 93.0 94.6 93.5 92.6 93.9 99.6 100
11 A. sydowii NRRL 254 93.2 93.5 94.5 93.0 94.1 93.2 91.9 93.4 98.3 98.0 100
12 A. versicolor NRRL 238 93.4 93.7 94.6 92.8 94.3 93.4 92.1 93.5 98.5 98.5 99.1 100
13 A. nidulans ATCC 10074 94.5 94.7 95.4 93.6 94.8 94.1 92.8 93.4 98.7 98.7 97.4 97.6 100
a
Values were calculated from pairwise alignments using sequence data determined in this study (for GenBank accession numbers, see Table 1) and the
STRETCHER algorithm. Species numbers were assigned arbitrarily.
b
Sequences were derived from type strains or from non-type strains (A. flavus ATCC 11497, A. niger ATCC 1015, and A. restrictus NRRL 148) that exhibited perfect
sequence identity with corresponding type strain reference data (35).

divergent among Aspergillus species in the ITS1 region than in species than between species and ranged from 1 nt (e.g., A.
the D1-D2 region; i.e., in the ITS1 region, 5 of the 13 species niger) to 3 nt (A. flavipes) (Table 1).
studied demonstrated a unique sequence length (A. candidus, Therefore, as noted for data generated using the D1-D2 and
A. chevalieri, A. granulosus, A. nidulans, and A. terreus) com- ITS1 regions, the overall sequence length of the ITS2 region
pared to only one species (A. nidulans) in the D1-D2 region. In alone could not be used reliably to differentiate among all 13
addition, the overall sequence length for the type strain of A. Aspergillus species examined. However, when data was com-
ustus (NRRL 275) not only was unique compared to other bined for both the ITS1 and ITS2 regions, including or exclud-
Aspergillus species but differed by 6 nt from its own sequevars ing the intervening, conserved, 5.8S rRNA gene, all 13 Aspergil-
(i.e., 161 nt versus 155 nt); these data suggest substantial di- lus type strain sequences could be differentiated from one
vergence of these sequevars from the type strain. However, all another (ITS1 plus ITS2 overall sequence length [Table 1]).
of the 7 remaining species and the two divergent A. ustus The only overlaps in overall sequence length were between A.
sequevars (I and II) shared identical overall sequence length candidus (505 nt) and one non-type strain sequevar of A. fla-
with at least one strain from each of the 13 species examined vipes (A. flavipes I; 505 nt) and between a non-type strain
(e.g., A. flavipes and A. niger, 185 nt [Table 1]). Identical or sequevar of A. nidulans (A. nidulans II, 480 nt) and A. sydowii.
similar overall sequence lengths were observed for A. candidus, Pairwise nucleotide sequence comparison of the D1-D2,
A. flavus, and A. restrictus (180 to 181 nt), A. flavipes, A. fu- ITS1, and ITS2 ribosomal regions among Aspergillus species
migatus, A. niger, and A. terreus (184 to 186 nt), and A. granu- investigated in this study. Table 2 shows the results of pairwise
losus, A. nidulans, A. sydowii, A. ustus (sequevars I and II), nucleotide sequence analysis of the D1-D2 region, and Tables
and A. versicolor (153 to 156 nt). In some instances, overall 3 and 4 show the same analysis but for the ITS1 and ITS2
sequence length differences varied more within a given species ribosomal regions. D1-D2 sequences were found to be highly
than between species and ranged from 1 nt (A. flavipes, A. conserved among the aspergilli (91.9 to 99.6% identity [Table
nidulans) to 6 nt (A. ustus) (Table 1). 2]). The two species with the greatest similarity in nucleotide
In comparison, ITS2 regions varied in overall length from sequence were A. ustus and A. granulosus (99.6% identity); A.
160 nt (A. flavipes) to 177 nt (A. terreus) (Table 1). Overall sydowii and A. versicolor sequences were also very similar and
sequence length was slightly less divergent among the Aspergil- shared 99.1% sequence identity. Those species that were the
lus species in the ITS2 than in the ITS1 region but was more most dissimilar in D1-D2 sequence were A. sydowii and A.
divergent than in the D1-D2 region (Table 1). Three of the 13 restrictus (91.9% sequence identity). Phylogenetically related
species examined demonstrated a unique overall sequence species, i.e., representatives of the Aspergillus subgenus Nidu-
length in the ITS2 region (A. chevalieri, A. granulosus, and A. lantes (35), demonstrated more than 97% sequence identity: A.
terreus), and 5 shared identical overall sequence length with at granulosus, 98.3 to 99.6% identity with A. ustus, A. sydowii, A.
least 1 strain from each of the 13 species examined (e.g., A. versicolor, and A. nidulans; A. ustus, 98.0 to 98.7% identity with
chevalieri and A. versicolor, 167 nt). Identical or similar se- A. sydowii, A. versicolor, and A. nidulans; and A. sydowii, 99.1%
quence lengths were observed for A. candidus, A. chevalieri, A. identity with A. versicolor. In addition, A. sydowii and A. versi-
flavus, A. fumigatus, A. nidulans, A. niger, A. sydowii, and A. color shared 97.4 and 97.6% sequence identity, respectively,
versicolor (167 to 169 nt) and among A. granulosus, A. restrictus, with A. nidulans.
and A. ustus (170 to 172 nt). In some instances, similar to the In contrast to the D1-D2 region, pairwise nucleotide se-
results obtained in the ITS1 analysis, overall sequence length quence analysis of the ITS1 region demonstrated significantly
differences in the ITS2 region varied more within a given more variation in nucleotide sequences among the aspergilli
2096 HINRIKSON ET AL. J. CLIN. MICROBIOL.

TABLE 3. Pairwise sequence comparison in ITS1 regions between medically important Aspergillus species investigated in this study

Species % Sequence identity with species no.a:

Species and strainb
no. 1 2 3 4 5 6 7 8 9 10 11 12 13

1 A. fumigatus ATCC 1022 100

2 A. flavus ATCC 11497 77.4 100
3 A. niger ATCC 1015 85.0 79.5 100
4 A. terreus ATCC 1012 84.0 78.0 87.8 100
5 A. flavipes ATCC 24487 79.5 75.3 82.3 86.8 100
6 A. candidus NRRL 303 84.0 82.3 82.4 79.8 78.7 100
7 A. restrictus NRRL 148 83.1 75.0 83.0 80.4 77.4 78.9 100
8 A. chevalieri ATCC 16443 63.8 60.0 62.6 60.4 57.8 61.5 71.6 100
9 A. granulosus NRRL 1932 58.9 61.2 58.2 58.5 60.6 60.1 57.4 63.8 100

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10 A. ustus NRRL 275 59.0 60.4 59.2 60.5 58.7 58.7 59.2 60.1 92.5 100
11 A. sydowii NRRL 254 62.6 61.8 60.7 59.5 59.5 63.2 61.7 68.5 73.9 72.6 100
12 A. versicolor NRRL 238 62.6 61.3 62.3 60.5 60.1 62.3 61.7 69.8 73.3 70.7 98.1 100
13 A. nidulans ATCC 10074 63.7 60.4 61.3 60.0 60.4 62.1 61.1 67.5 74.8 75.0 94.2 94.8 100
a
Values were calculated from pairwise alignments using sequence data determined in this study (for GenBank accession numbers, see Table 1) and the
STRETCHER algorithm. Species numbers were assigned arbitrarily.
b
Sequences were derived from type strains or from non-type strains (A. flavus ATCC 11497, A. niger ATCC 1015, and A. restrictus NRRL 148) that exhibited perfect
sequence identity with corresponding type strain reference data (15).

(Table 3). Sequence identities among the 13 Aspergillus species all belonging to the subgeneric Nidulantes group, demon-
ranged from 57.4 to 98.1%. The two species with the greatest strated sequence identities between 93% and 98.3%: A. granu-
similarity in nucleotide sequence were A. sydowii and A. versi- losus (93.6 to 98.3% identity with A. versicolor, A. sydowii, A.
color (98.1% identity) and those that were the most dissimilar nidulans, and A. ustus), A. ustus (94.8% identity with A. nidu-
were A. granulosus and A. restrictus (57.4% identity). Only four lans), A. sydowii (95.9 and 97% identity with A. nidulans and A.
pairs of species, all belonging to the subgeneric Nidulantes versicolor, respectively), and A. nidulans (95.3% identity with
group, demonstrated sequence identities greater than 92% (A. A. versicolor).
ustus versus A. granulosus, 92.5% identity; A. versicolor versus Alignment of DNA sequences of the D1-D2, ITS1, and ITS2
A. sydowii, 98.1% identity; A. nidulans versus A. sydowii or A. ribosomal regions among Aspergillus species investigated in
versicolor, 94.2 and 94.8% identity, respectively). this study. DNA sequence alignments of the D1 and D2 re-
On the other hand, pairwise nucleotide sequence analysis of gions and of the ITS1 and ITS2 regions were conducted to
the ITS2 region demonstrated intermediate variation among identify areas within each region which displayed the greatest
Aspergillus species compared to the D1-D2 and ITS1 regions diversity and which might best discriminate among the As-
(Table 4). Sequence identities among the 13 Aspergillus species pergillus species examined. Although some interspecies se-
ranged from 75.6 to 98.3% identity. The two species with the quence divergence was observed in the more conserved D1
greatest similarity in nucleotide sequence were A. granulosus region, the most significant sequence divergence among As-
and A. ustus (98.3% identity), agreeing with the D1-D2 simi- pergillus species occurred in the more variable D2 region (i.e.,
larity ranking; those that were the most dissimilar were A. in the D1 region, 27 [19.7%] divergent sites occurred over 137
terreus and A. restrictus (75.6% identity). Eight pairs of species, aligned positions; in the D2 region, 55 [26.3%] divergent sites

TABLE 4. Pairwise sequence comparison in ITS2 regions between medically important Aspergillus species investigated in this study

Species % Sequence identity with species no.a:

Species and strainb
no. 1 2 3 4 5 6 7 8 9 10 11 12 13

1 A. fumigatus ATCC 1022 100

2 A. flavus ATCC 11497 87.7 100
3 A. niger ATCC 1015 87.1 87.2 100
4 A. terreus ATCC 1012 85.9 83.3 83.6 100
5 A. flavipes ATCC 24487 87.5 85.9 83.4 83.6 100
6 A. candidus NRRL 303 89.4 86.0 85.9 84.2 86.3 100
7 A. restrictus NRRL 148 81.5 80.0 79.5 75.6 78.0 83.3 100
8 A. chevalieri ATCC 16443 80.1 80.9 75.9 76.4 76.6 82.5 83.2 100
9 A. granulosus NRRL 1932 84.6 83.4 84.7 83.5 86.6 85.2 78.1 79.2 100
10 A. ustus NRRL 275 85.3 83.6 84.1 82.3 85.5 84.2 78.8 78.7 98.3 100
11 A. sydowii NRRL 254 85.1 83.8 85.5 82.2 87.0 86.1 78.5 78.8 94.2 91.9 100
12 A. versicolor NRRL 238 86.7 84.9 85.6 81.8 88.2 86.2 79.0 78.8 93.6 91.9 97.0 100
13 A. nidulans ATCC 10074 87.2 82.1 85.0 82.7 86.9 85.0 79.5 79.4 95.3 94.8 95.9 95.3 100
a
Values were calculated from pairwise alignments using sequence data determined in this study (for GenBank accession numbers, see Table 1) and the
STRETCHER algorithm. Species numbers were assigned arbitrarily.
b
Sequences were derived from type strains or from non-type strains (A. flavus ATCC 11497, A. niger ATCC 1015, and A. restrictus NRRL 148) that exhibited perfect
sequence identity with corresponding type strain reference data (15).
VOL. 43, 2005 RIBOSOMAL IDENTIFICATION OF ASPERGILLUS SPECIES 2097

occurred over 209 aligned positions) (Fig. 1). Closely related A. ustus) were found to be identical with, or very similar to,
taxa differed by ⱕ3 nt in the D2 region, e.g., A. granulosus reference sequences from closely related but different taxa
versus A. ustus (1 nt) and A. sydowii versus A. versicolor (3 nt). (molecular siblings) (Table 5). For instance, A. flavus D1-D2
Intraspecies sequence variability was ⱕ2 nt over the entire sequences were not distinguished from sequences representing
D1-D2 region (A. flavipes, A. sydowii, and A. nidulans differed Aspergillus oryzae, Aspergillus parasiticus, Aspergillus sojae,
by 1 to 2 nt from their respective type strains; remaining spe- Aspergillus subolivaceus, or Aspergillus terricola (100% identity
cies differed by 0 nt). in the D1-D2 region, Table 5). Only 1 nt difference (99.8%
In comparison, Fig. 2 shows DNA sequence alignments for identity) was observed between our D1-D2 sequences for
the ITS1 and ITS2 regions of the Aspergillus strains investi- strains of A. flavus and GenBank entries for Aspergillus tamari
gated. In contrast to the D1-D2 regions, the ITS1 sequence and Aspergillus flavofurcatus (Table 5). In total, 8 of the 13
alignment revealed five regions with significant interspecies Aspergillus species examined demonstrated identical D1-D2
divergence (ITS1 variable regions 1 to 5) (Fig. 2). The diversity sequences with at least one molecular sibling and the remain-

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within these regions was the result of nucleotide polymor- der of the species gave sequence identities of 99.3 to 99.8%
phisms as well as differences in sequence length between some with molecular siblings (Table 5).
groups of species studied. The most prominent difference in All BLAST results, using our own ITS1 and ITS2 data as the
sequence length was noted in ITS1 variable region 3, which query sequences, gave top-ranking scores (100% sequence
exhibited a significant deletion (36 to 49 aligned positions) in identity) with corresponding GenBank reference sequences;
the strains representing A. chevalieri and the subgeneric group the only exception was for A. granulosus because no sequences
Nidulantes (A. nidulans, A. versicolor, A. sydowii, A. ustus, and for the ITS regions were available for this species at the time
A. granulosus). In addition, signature nucleotide sequence pat- of our study. Although our sequences usually showed 100%
terns could be discerned among closely related species within identity with GenBank reference data for Aspergillus type
each of the five variable regions and throughout the remaining strains, if available (15), some variations were detected among
parts of the ITS1 alignment; e.g., among A. granulosus and A. sequevars; however, these differences did not change the spe-
ustus, and among A. sydowii and A. versicolor (Fig. 2). Intraspe- cies identification of a given sequevar based on GenBank
cies sequence variability, if present, was ⱕ3 nt (A. flavipes: 3 nt, search data nor did it alter the overall composition of top-
⬍2%; A. niger: 3 nt, ⬍2%; A. nidulans: 2 nt, ⬍2%) except for ranking matches, i.e., the list of molecular siblings. Therefore,
A. ustus strains that differed from the type strain sequence by BLAST results for sequevars other than those representing
11 to 14 aligned positions in the ITS1 region. type strains were not included in Table 5.
Alignment of ITS2 sequences revealed significant interspe- Unlike GenBank search results using query sequences from
cies divergence in the area proximal to the 5⬘ end (ITS2 vari- the D1-D2 region, where 8 of the 13 species gave 100% iden-
able region 1) and at the 3⬘ end (ITS2 variable region 2) (Fig. tical sequence data for at least one molecular sibling and all
2). These regions varied more in nucleotide polymorphisms remaining species gave sequence similarities of 99.3% to
than in sequence length, in contrast to the ITS1 variable re- 99.8% with their respective molecular siblings (Table 5), ITS1
gions; however, both ITS2 variable regions demonstrated sig- sequences gave 100% identity with molecular siblings for only
nificant sequence length differences between unrelated species 4 of the 13 Aspergillus species studied (A. chevalieri, A. flavus,
compared to related species. As noted for the ITS1 region, A. nidulans, and A. niger); the remaining species gave sequence
signature nucleotide sequence patterns could be identified similarities ranging from 84.3% (A. granulosus) to 99.4% (A.
among closely related species throughout the ITS2 region, restrictus) with sibling species (Table 5). ITS2 sequences gave
especially within its variable region 2 (Fig. 2). Intraspecies 100% identity with molecular siblings for only 5 of the 13
nucleotide variability, if present, was ⱕ5 nt (A. ustus, 5 nt, Aspergillus species studied (A. chevalieri, A. flavus, A. nidulans,
⬍3%; A. nidulans and A. niger, 1 nt, ⬍1%; remaining species, A. niger and A. ustus); the remaining species gave sequence
0 nt), except for A. flavipes strains that differed from the type similarities ranging from 91.5% (A. terreus) to 99.4% (A. niger)
strain sequence by 4 to 8 aligned nucleotides in the ITS2 with sibling species (Table 5). However, it must be noted that
region. ITS reference data were not available in GenBank for all
Comparative GenBank analysis of D1-D2, ITS1, and ITS2 sibling species, especially for Eurotium, Emericella, and Neo-
ribosomal sequences. To determine the usefulness of a public sartorya species, at the time of our study.
database to serve as a guide to species identification by com- Assessment of the quality of D1-D2, ITS1 and ITS2 se-
parative DNA sequence analysis, D1-D2, ITS1, and ITS2 se- quence entries in GenBank. Inspection of BLAST alignments,
quences for each strain of the 13 Aspergillus species studied and of multiple alignments generated with Aspergillus species
were used to conduct BLAST searches of the GenBank data- D1-D2, ITS1, and ITS2 data from GenBank, revealed that
base (Table 5). All BLAST search results, using our own many Aspergillus species sequences in this database had trun-
D1-D2 data as the query sequences, gave top-ranking scores cated ends and/or heterogeneities at positions found to be
(100% sequence identity) with corresponding reference se- conserved at the generic or subgeneric level among the refer-
quences (35). GenBank searches revealed that sequences for ence sequences of type strains and of authenticated culture
strains of A. flavipes, A. sydowii, and A. versicolor were clearly collection strains (15, 35). These data indicate that many of the
distinct from those of other Aspergillus species or related taxa current GenBank sequences are not complete and/or contain
(i.e., ⱖ4 nt or ⱕ99.3% similarity with any other GenBank entry errors in sequence data for the ribosomal regions studied.
for a different species); however, sequences for the remaining Further, BLAST search analysis of some query sequences gave
species (A. candidus, A. chevalieri, A. flavus, A. fumigatus, A. best-match results with reference sequences of corresponding
granulosus, A. nidulans, A. niger, A. restrictus, A. terreus, and type strains but also resulted in excellent scores for sequences
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J. CLIN. MICROBIOL.
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2098
VOL. 43, 2005 RIBOSOMAL IDENTIFICATION OF ASPERGILLUS SPECIES 2099

assigned to (i) unrelated or distantly related taxa, i.e., Cordyceps conclusive at the species level due to identical or very similar
sinensis AJ488259, AJ488260, and AJ488268 (A. chevalieri (⬎99% identical) reference data which were assigned to other
query), Aureobasidium mansonii AF121288 (A. flavus organisms than the query sequences (molecular siblings).
query), and A. wentii U03522 and U03523, Arthrobotrys spe- Nonetheless, these sibling organisms most frequently belonged
cies U72602, Gliocladium cibotii AF021264, AF048739, and to the same Aspergillus group as the query species (35) and
AF048738, and Verticillium bulbillosum AF048741 (A. niger supported the identification to at least the subgeneric group
query), or (ii) more closely related but different taxa, i.e., level. Consequently, results obtained by sequencing the D1-D2
A. nidulans AF455499 (A. sydowii query), A. nidulans region should be interpreted accordingly. The same test spec-
AF455424, AF455505, and AY452983 (A. versicolor query), ificity applies to currently available large-subunit rRNA gene
and A. sydowii AJ312221 (A. nidulans query). When reference probes (42) which target D2 sequences conserved among med-
alignments were used for type strains, all of these GenBank ically important species and their molecular siblings. In addi-
entries were shown to exhibit significant nucleotide differences tion, other targets with conserved interspecies sequences are

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compared to respective reference sequences. These results in- expected to exhibit analogous diagnostic limitations, such as
dicate the existence of mislabeled ribosomal Aspergillus species those reported previously using the mitochondrial cytochrome
data in GenBank (“false” molecular siblings). We could not b gene (46, 58).
determine, however, whether these ambiguous sequence an- Unlike the D1-D2 region, the two ITS regions investigated
notations were due to misnamed or misidentified isolates or to were found to exhibit localized hypervariable regions contain-
manipulation or editing errors occurring during sequence anal- ing most of the intraspecies sequence diversity. Nonetheless,
ysis or GenBank submission. most ITS amplicons showed a consistent sequence length
among strains of the same species in one or both spacer re-
DISCUSSION gions; exceptions included sequevars of A. flavipes, A. nidulans,
A. niger, and A. ustus. These hypervariable regions also con-
The increasing popularity of molecular approaches for the tained most of the interspecies sequence diversity; however,
identification of fungal pathogens reflects significant improve- similar sequence lengths in one or both spacer regions often
ments in DNA analysis in recent years, including the develop- occurred, making the overall size of ITS amplicons an imper-
ment of broad-range (panfungal) primers, kit-based automated fect tool to differentiate medically important Aspergillus spe-
sample processing and DNA amplification systems, and ex- cies. This was especially evident when data from only one
panding public and private DNA sequence databases. The spacer region were analyzed. It is also not known if sibling
most promising results were derived from ribosomal sequenc- species or other potential pathogens possess similar amplicon
ing studies examining yeasts and dermatophytes; however, di- lengths that would confound a specific identification because,
morphic fungi and moulds are also being investigated in order at present, GenBank data are incomplete in the ITS region.
to overcome diagnostic problems encountered by extended Our work therefore supports a previous report suggesting that
incubation periods and the plasticity of traditional identifica- A. fumigatus and A. flavus could not be differentiated by ITS
tion criteria (5, 13, 14, 18, 27, 28, 40). An important prerequi- amplicon length alone and that application of a specific DNA
site for the development of clinical molecular approaches is, of capture probe may be required for species identification (16).
course, the estimation of the target resolution at the species Alternatively, ITS amplicons have been characterized using
level using a comprehensive collection of reference strain se- single-strand conformation polymorphism (SSCP) analysis ex-
quences. Unfortunately, such an assessment has not been pub- ploiting both size and sequence differences (23, 56); however,
lished to date for the various ribosomal targets of Aspergillus clinical diagnostic applications may be compromised by in-
species. Therefore, we evaluated the utility of the ribosomal traspecies SSCP pattern variability such as that described re-
D1-D2, ITS1, and ITS2 sequences as targets for the differen- cently for strains of A. fumigatus and A. flavus (39). Multiple
tiation of medically important Aspergillus species by compre- SSCP patterns are also predicted to occur within species of A.
hensive DNA sequence analysis of type cultures and authen- flavipes, A. nidulans, A. niger, A. ustus, and other Aspergillus
ticated culture collection reference strains and conducted a species that exhibit sequence differences among strains of the
systematic comparison of these sequences to those available in same species (this study; 15, 34, 44, 53, 54).
the GenBank database. The presence of intraspecies ITS variability did not hamper
Primary structural analysis of our Aspergillus species D1-D2 Aspergillus species identification by comparative sequence
sequences revealed negligible intraspecies variability and rec- analysis, as indicated by similar BLAST search results for all
ognizable interspecies divergence within the D2 region as de- sequevars of a particular species. All ITS sequences deter-
scribed for other fungi (12, 24, 25, 36, 42). Although compar- mined in this study yielded top-ranking BLAST scores with
ison of the D1-D2 sequences determined in this study with corresponding reference data (15, 34) regardless of the spacer
those in GenBank showed complete identity with correspond- region analyzed (ITS1 or ITS2) except for A. granulosus be-
ing reference data (35), most BLAST search results were in- cause its ITS sequence was not available in GenBank at the

FIG. 1. Alignment of complete Aspergillus D1-D2 regions illustrating the sequence divergence among medically important species. The highly
conserved region of the 28S rRNA gene intervening between the D1 and D2 regions has been omitted. Dots indicate identical nucleotides
compared to the leader sequence (A. fumigatus strain ATCC 1022); dashes indicate alignment gaps. Sequence data were derived from this study
(for GenBank accession numbers, see Table 1).
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J. CLIN. MICROBIOL.
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VOL. 43, 2005 RIBOSOMAL IDENTIFICATION OF ASPERGILLUS SPECIES 2101

TABLE 5. Molecular siblings of medically important Aspergillus species investigated in this study
Maximum sequence identity (%) per target
Query speciesa Molecular siblingb (minimum nucleotide position difference)c

D1-D2 ITS1 ITS2

d
A. candidus A. campestris 100 (0) NA NA
e
A. (Eurotium) chevalieri Various Eurotium species 100 (0) 100 (0) 100 (0)

A. (Fennellia) flavipes None

A. flavus A. oryzae 100 (0) 100 (0) 100 (0)

A. parasiticus 100 (0) 97.8 (4) 97.7 (4)

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A. sojae 100 (0) 97.8 (4) 97.7 (4)
A. tamarii 99.8 (1) 95.6 (8) 97.0 (5)
A. subolivaceus 100 (0) NA NA
A. terricola 100 (0) NA NA
A. flavofurcatus 99.8 (1) NA NA

A. fumigatus Various Neosartorya speciese 99.8 (1) 98.9 (2) 97.0 (5)

A. granulosus A. ustus 99.8 (1) 94.3 (9) 98.3 (3)

A. puniceus 99.6 (2) 84.3 (27) 98.3 (3)
A. pseudodeflectus 99.8 (1) NA NA

A. (Emericella) nidulans Various Emericella speciese 100 (0) 100 (0) 100 (0)

A. niger A. awamori NA 100 (0) 100 (0)

A. foetidus NA 100 (0) 100 (0)
A. phoenicis 100 (0) 98.9 (2) 99.4 (1)
A. restrictus A. caesiellus 99.8 (1) 99.4 (1) 96.5 (6)
A. conicus 100 (0) NA NA

A. sydowii A. versicolor 99.1 (5) 98.1 (3) 98.2 (3)

A. terreus Fennellia nivea 100 (0) 92.6 (14) 91.5 (15)

A. carneus 100 (0) NA NA
A. allahabadii 99.8 (1) NA NA

A. ustus A. granulosus 99.6 (2) 92.5 (12) 98.3 (3)

A. puniceus 100 (0) 89.5 (18) 100 (0)
A. pseudodeflectus 99.8 (1) NA NA

A. versicolor A. sydowii 99.3 (4) 98.1 (3) 98.8 (2)

a
See Table 1 for the origin of strains used and for the corresponding GenBank sequence accession numbers; sequence data for only the type strains or reference
strains with identical ribosomal sequences compared to corresponding type strains are shown for ease of presentation because despite some nucleotide variations among
sequevars of a given species, molecular siblings captured from GenBank were the same for all sequevars within a given species (see Results section).
b
Organism(s) assigned to a different species than query sequence although exhibiting identical or very similar (⬎99% identity) sequences in at least one ribosomal
region investigated in this study. The listing is limited to Aspergillus species currently sanctioned by the International Commission of Penicillium and Aspergillus (37).
Note that the query sequences of A. chevalieri, A. flavus, A. nidulans, A. niger, A. sydowii, and A. versicolor also revealed “false” molecular siblings in GenBank, i.e.,
GenBank sequences with ambiguous classification as detailed in the Results section of this study.
c
Comparison between query sequences (this study) and reference sequences of molecular siblings obtained from GenBank (15, 35) and the present investigation (A.
granulosus ITS sequences) using BLAST and GCG algorithms.
d
NA, no corresponding reference data available for this molecular sibling in this target region at the time this study was conducted.
e
Query sequences matched multiple species of this genera in the D1-D2 and ITS regions although ITS reference data for many species of this taxonomic group were
not available in GenBank at the time this study was conducted.

time of this study. Reference sequences of organisms most existed for different organisms (molecular siblings of A. cheva-
closely related to, but different from, the query species usually lieri, A. flavus, A. nidulans, and A. niger). Additional ambiguity
exhibited less than 99% sequence identity in at least one spacer arose from the existence of probably misclassified or misnamed
region. Nonetheless, some ITS analyses were inconclusive at GenBank sequences exhibiting excellent BLAST scores with
the species level because similar GenBank reference sequences some of our query sequences (A. chevalieri, A. flavus, A. nidu-

FIG. 2. Alignment of complete Aspergillus ITS1-ITS2 regions illustrating the sequence divergence among medically important species. The
highly conserved intervening 5.8S rRNA gene has been omitted. Dots symbolize identical nucleotides compared to the leader sequence (A.
fumigatus strain ATCC 1022); dashes indicate alignment gaps. Sequence data were derived from this study (for GenBank accession numbers, see
Table 1).
2102 HINRIKSON ET AL. J. CLIN. MICROBIOL.

lans, A. niger, A. sydowii, and A. versicolor) and yet assigned to investigated in this study can largely be identified at the tradi-
different taxa (including Arthrobotrys, Aspergillus, Aureobasi- tional group level using ribosomal D1-D2 or ITS regions as
dium, Cordyceps, Gliocladium, and Verticillium species— diagnostic targets. Upon major improvement of present se-
“false” molecular siblings [see Results]). Truncated and low- quence databases, identification to the species level should be
quality GenBank sequences were disregarded in the present feasible through ITS sequence analysis; some clinical isolates,
study and therefore did not affect identification; however, trun- however, may require additional analyses. Such improved da-
cated and low-quality GenBank entries clearly limit the utility tabases should enable a more accurate identification of As-
of this database to assess inter- and intraspecies sequence pergillus species, thus promoting further advancements in the
similarity. Overall, ITS comparative sequence analysis, which molecular diagnosis, epidemiology, and clinical management
included both spacer regions, showed better species differen- of invasive aspergillosis.
tiation than use of a single spacer region alone. However,
differentiation between some more closely related Aspergillus ACKNOWLEDGMENTS

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species, especially among Eurotium and Emericella species, This work was supported by a postdoctoral grant (no. 81ZH-59638)
may require analysis of less conserved targets such as the from the Swiss National Science Foundation to H.P.H.
ribosomal external transcribed spacer regions or intergenic We thank Stephen W. Peterson and Arvind A. Padhye for kindly
spacer regions (18). providing reference strains and helpful advice concerning the taxon-
omy of Aspergillus species. Brandi Reddick is acknowledged for excel-
Considering the results presented above, the development of lent technical assistance.
ribosomal approaches targeting Aspergillus groups rather than
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