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Journal of Clinical Microbiology, June 1999, p. 1846-1851, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid Identification of Fungi by Using the ITS2 Genetic Region
and an Automated Fluorescent Capillary Electrophoresis System
Christine Y.
Turenne,1,2,*
Steven E.
Sanche,3
Daryl J.
Hoban,1,4
James A.
Karlowsky,1,2,4 and
Amin M.
Kabani1,2,5
Department of Medical Microbiology, Faculty
of Medicine,1 and Faculty of
Pharmacy,4 University of Manitoba, and
Departments of Clinical Microbiology2
and Medicine,5 Health Sciences Centre,
Winnipeg, Manitoba R3A 1R9, Canada, and Division of
Infectious Diseases, Royal University Hospital, Saskatoon,
Saskatchewan,3 Canada
Received 28 December 1998/Returned for modification 26 January
1999/Accepted 17 March 1999
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ABSTRACT |
Invasive fungal disease often plays an important role in the
morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in
rapid and accurate diagnosis of clinical fungal isolates. PCR with
fungus-specific primers targeted toward conserved sequences of the 5.8S
and 28S ribosomal DNA (rDNA) results in amplification of the
species-specific ITS2 regions, which are variable in amplicon length.
We have made use of the ABI PRISM 310 genetic analyzer and the ABI
PRISM 310 GeneScan analysis software for the determination of variable
size differences of the ITS2 region of clinically important fungi,
including Candida and non-Candida yeasts,
Aspergillus species, and a variety of dermatophytes. No
cross-reaction occurred when samples were tested against human and
bacterial genomic DNA. We have found that most clinically significant
fungal isolates can be differentiated by this method, and it therefore
serves to be a promising tool for the rapid (<7 h) diagnosis of
fungemia and other invasive fungal infections.
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INTRODUCTION |
Advances in medicine contributing to
the increased survival of immunocompromised patients, including
oncology, human immunodeficiency virus-infected, diabetes, and
transplant patients, have also brought forth an increase in the
prevalence of nosocomial fungal infections (1, 2, 11). These
infections carry a high mortality, ranging from 30 to 60% (2, 9,
11, 18), depending on the underlying condition and whether
effective antifungal therapy was administered. Tissue involvement can
occur in up to 36% of fungemic episodes, which has been associated
with an even higher mortality rate of 47 to 88% (8, 9, 28).
Disseminated infections due to some organisms such as
Aspergillus and Fusarium species have a mortality rate close to 100% (9, 18).
Candida species now rank fourth among the most commonly
isolated organisms from bloodstream infections (1, 3, 19). There has also been a rise in the incidence of disease caused by
non-albicans Candida species (17, 20). While
Candida albicans (~60%) and Candida species
(~20%) are responsible for most fungal infections (2, 9),
up to 150 fungal species have been demonstrated to be primary pathogens
of humans, involving all body sites (6). Furthermore, this
problem is compounded by an increase in resistance to antifungal
agents, particularly the azoles (20, 21) and amphotericin B
(17), and an increase in the empirical use of these agents.
Early detection of infection has a great impact on the clinical outcome
of many infectious diseases. Unfortunately, the identification of fungi
by traditional morphologic and metabolic characteristics may take days
to weeks. For molds in particular, these methods are laborious,
time-consuming, and require significant technological expertise. Blood
culture systems may fail to detect as many as 45 to 75% of cases of
disseminated candidiasis (9, 23) and most cases of invasive
aspergillosis (25). Consequently, when a blood culture
result is positive for pathogenic and opportunistic fungi, far too
often it is obtained just prior to death, when it is too late.
Therefore, a high index of suspicion is required, leading to the
empiric use of antifungal therapy. While the choice of treatment is
speculative, based on the most probably pathogens involved, the
standard choice of antifungal treatment often remains amphotericin B. However, as more alternative antifungal agents with various spectra of
activity are being developed, specific identification of pathogenic
fungi will become even more important in the near future. Investigators
have attempted to overcome these difficulties by developing rapid,
sensitive detection and identification methods with the intent of
improving patient outcome and reducing costs.
At the molecular level, genetic sequence variation offers an
alternative to culturing for detection and identification of fungi. For
example, the ribosomal genes demonstrate conserved sequence regions
ideal for primer targeting as well as regions of variability useful for
species identification. Amplification techniques, with subsequent
probing of the amplicons with species-specific probes or in a
PCR-enzyme immunoassay format, have been utilized to overcome the
problems of sensitivity, specificity, and delay encountered with
conventional methodology (4, 5, 7, 10, 22, 24, 29). These
methods have already shown great promise in the field of diagnostics.
However, the use of species-specific probes is not always an efficient
approach in mycology, given the large number of potentially pathogenic fungi.
PCR primers that target conserved regions of fungal rRNA genes,
amplifying sequence-variable fragments of genes or intervening noncoding regions (26), have been used for sequence
comparisons for phylogenetic analyses of a variety of fungal groups.
Interspecies variability is also manifested in the fragment size of the
internal transcribed spacer 1 and 2 (ITS1 and ITS2, respectively)
regions (15, 27). We have utilized the variability in length
of the ITS2 region to make specific diagnosis of pathogenic fungal
isolates from blood and tissues (Fig. 1).
This is a promising method; however, the size differences of the
amplicons from different species may not be detectable by agarose gel
electrophoresis. In this study, we used the ABI PRISM 310 genetic
analyzer, an automated capillary electrophoresis sequencer, to detect
fluorescently tagged amplicons from the ITS2 region. The basic method
for this application is described as follows. A single capillary is
filled with a replaceable liquid polymer for high resolution under
denaturing conditions between each sample test. The sample containing
fluorescent PCR product is injected into the capillary and is subject
to constant voltage and temperature (60°C). The fluorescently tagged
fragments of DNA resulting from amplification by fluorescent primers
are detected by a laser beam and automatically analyzed by the
instrument. Between 1 and 96 samples can be loaded at any one time on
the instrument, and each run is completed within 30 min. Results can be
viewed as soon as each sample has been processed. The instrument detects up to four various fluorescent "colors," allowing for the
inclusion of an internal standard with each run. Accurate fragment size
analysis based on the electrophoretic mobility of the sample relative
to the internal standard is determined by using the ABI PRISM 310 GeneScan analysis software. This method has enabled us to generate a
profile of expected amplicon sizes for a broad range of yeasts and
molds, allowing for their rapid identification.
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FIG. 1.
Schematic representation of the fungal ribosomal genes
containing the primer target areas used in the amplification of the
ITS2 region.
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MATERIALS AND METHODS |
Fungal strains.
For the testing of primer specificity, a
reference strain of Candida albicans (ATCC 10231) was used.
For the determination of the ITS2 PCR fragment length, a wide
collection of reference strains, American Type Culture Collection or
College of American Pathologist specimens were used, as well as patient
strains obtained from the Health Sciences Centre Clinical Microbiology
Laboratory stock culture collection. Yeast organisms were grown on
Sabouraud dextrose agar plates (BBL, Becton-Dickinson, Cockeysville,
Md.) for 24 h at 37°C, and molds were grown on potato dextrose
agar (16) for up to 7 days. Species identification was
established by using the API 20C kit (BioMerieux, Hazelwood, Mo.) or by
conventional morphological analysis (14).
DNA isolation from molds.
Mold scrapings were suspended in 1 ml of T10E1 buffer (10 mM Tris-HCl, 1 mM EDTA
[pH 8.0]) followed by centrifugation at 13,000 rpm for 5 min. The
pellet was resuspended in 200 µl of 50 mM NaOH, vortexed, and
incubated at 95°C for 10 min. The mixture was then neutralized with
200 µl of 0.1 mM Tris-HCl (pH 7.0) and centrifuged for 5 min at
13,000 rpm. The pellet was resuspended in 500 µl of sterile
H2O and centrifuged at 13,000 rpm for 5 min. The
supernatant was removed, followed by the addition of 200 ml of yeast
cracking buffer (2% Triton X-100, 1% sodium dodecyl sulfate, 100 mM
NaCl, 20 mM Tris [pH 8.0], 10 mM EDTA [pH 8.0]). Extraction of
genomic DNA was achieved by addition of glass beads to 3/4 of the
liquid volume followed by the addition of 200 ml of cold
phenol-chloroform-isoamyl alcohol in a 25:24:1 ratio. The mixture was
subjected to constant vortexing for 30 min. The sample was then
centrifuged at 13,000 rpm for 5 min. The aqueous (top) layer was
transferred into a new tube, and 1 ml of cold (
20°C) 100% ethanol
was added for DNA precipitation. The sample was centrifuged at 13,000 rpm for 2 min. The supernatant was removed, and the pellet was
resuspended in 400 µl of T10E1 buffer and 30 µg of RNase A (Sigma, St. Louis, Mo.) for a 1-h water bath incubation
at 35°C. We then added 10 µl of 3 M sodium acetate and 1,000 µl
of cold 100% ethanol. The sample was centrifuged at 13,000 rpm for 2 min, the supernatant was removed, and the pellet was air dried. The DNA
pellet was resuspended in 50 µl of TE buffer, and was used as DNA
template for amplification after 30 min or stored at 20°C for
future use. This method, which can be used with all fungi we have
tested, is necessary for the extraction of DNA from certain molds, such as Aspergillus terreus, Penicillium sp.,
Paecilomyces sp., and Sporothrix schenckii.
DNA isolation from yeast.
Steps in the method described
above were omitted or reduced in duration if the organism was a yeast.
Resuspension and incubation of the fungal pellet in 50 mM NaOH were not
necessary. The vortexing with glass beads was reduced to three times
for 30 s, and the mixture was held on ice for short periods in
between. Finally, the RNase A incubation period was reduced to 5 min.
PCR amplification.
The primers used for universal fungal
amplification were ITS4 (reverse primer [5'-tcc tcc gct tat tga tag
c-3']) obtained from White et al. and fluorescently labeled (5'HEX)
ITS86 (forward primer [5'-gtg aat cat cga atc ttt gaa c-3']) (Life
Technologies, Burlington, Ontario, Canada) derived from sequence
comparison of various fungi from GenBank databases by using PCGene
software (University of Geneva, Geneva, Switzerland). The 50-µl PCR
reaction mixture contained 5 µl of DNA template; 5 µl of 25 mM
MgCl2-10× PCR buffer; 1.25 mM deoxynucleoside
triphosphate; dATP, dGTP, dCTP, and an 8:1 ratio of dUTP to dTTP; 0.5 µl of 100 µM each primer; 0.5 U of uracil DNA glycosylase (UDG)
(Gibco BRL); 2.5 U of Taq DNA polymerase (Pharmacia Biotech,
Baie d'Urfé, Québec, Canada); and 30 ml of sterile
distilled H2O. The PCR was performed in a GeneAmp PCR
system 9600 (Perkin-Elmer Applied Biosystems, Foster City, Calif.) with
cycles of 37°C for 10 min (UDG activation) and 94°C for 10 min (UDG
inactivation) and 30 cycles of 94, 55, and 72°C for 1 min each, and
then the mixture was incubated at 72°C for 10 min for final
extension. Amplification of all fungi tested with these primers yielded
fragments of 200 to 500 bp in length.
Primer specificity.
A variety or bacterial organisms were
used to determine the specificity of the fungal primers:
Staphylococcus epidermidis (ATCC 12228), Escherichia
coli (ATCC 25922), Staphylococcus aureus (ATCC 25923),
Pseudomonas aeruginosa (ATCC 27853), and Clostridium perfringens (ATCC 13124). Bacterial DNA was isolated by using the
QIAamp tissue kit (Qiagen, Santa Clarita, Calif.). The primers were
also tested against genomic DNA deriving from human whole blood,
concentrated leukocytes, and liver tissue. The PCR conditions were as
described above.
Agarose gel electrophoresis.
Detection of PCR-amplified
product was performed by electrophoresis on a 2% (wt/vol) agarose gel
stained with ethidium bromide. A volume of 20 µl of PCR product and
2.2 µl of Ficoll dye was loaded in each lane. Electrophoretic
conditions were 100 V for 45 min in 0.5× Tris-borate-EDTA buffer. A
123-bp ladder was also run in parallel for approximate PCR product band sizing.
Fragment analysis of the ITS2 region.
The ABI PRISM 310 genetic analyzer was used for determination of the precise lengths of
the PCR fragments containing the ITS2 regions. Fragment analysis was
done by using the ABI PRISM 310 GeneScan analysis software
(Perkin-Elmer Applied Biosystems). Sample preparation for capillary
electrophoresis involved the addition of 1 µl of diluted PCR product
(approximately 5 to 20 ng) to the capillary electrophoresis mix (12 µl of deionized formamide and 0.5 µl of GeneScan-500 (ROX) size
standard (Perkin-Elmer Applied Biosystems). A dilution of the PCR
product in TE buffer to a concentration of 5 to 20 ng/µl resulted in
an ideal peak intensity analyzable on the instrument relative to the
internal standard peaks. The capillary sample mix was denatured for 2 min at 95°C and rapidly cooled on ice prior to loading the
instrument. Subsequent preparation such as the setup of the ABI PRISM
310 genetic analyzer was done according to the manufacturer's
instructions with reference to using Performance Optimized Polymer 4 (POP-4) for microsatellite analysis (Perkin-Elmer Applied Biosystems).
A capillary (47 cm by 50 µm inside diameter) was installed, and POP-4
was used as the replaceable liquid polymer matrix. Electrophoresis
conditions were set on the instrument at a 5-s injection time, 15-kV
injection voltage, 15-kV electrophoresis voltage, 150-s syringe pump
time, 120-s preinjection electrophoresis, temperature constant of
60°C, and a 28-min collection time to ensure the detection of PCR
fragments under 450 bp in length. The matrix file (for POP-4, filter
set A, under the conditions described above) was created according to
the manufacturer's instructions to account for spectral overlap of the
various fluorescent molecules under the specific conditions used for
this application. Successful analysis was derived from the red peaks
being assigned to the ROX-labeled internal standard, for which the user
indicated in the program the appropriate fragment lengths, with the
green peak being assigned to HEX-labeled PCR fragments from fungal ITS2 amplification.
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RESULTS |
Primer specificity.
No amplification product was detected by
using the ITS86 and ITS4 primers with template from human leukocytes,
human whole blood, or human liver or against any of the following
bacterial organisms: Staphylococcus epidermidis,
Escherichia coli, Staphylococcus aureus,
Pseudomonas aeruginosa, and Bacteroides
fragilis (Fig. 2).
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FIG. 2.
Specificity of universal ITS2 primers against bacteria
and human genomic DNA. PCR amplification using the ITS4 and ITS86
primer pair was performed as described in Materials and Methods. The
following DNA templates were used for PCR (by lane): 1, Staphylococcus epidermidis ATCC 12228; 2, Escherichia
coli ATCC 25922; 3, Staphylococcus aureus ATCC 25923;
4, Pseudomonas aeruginosa ATCC 27853; 5, Clostridium
perfringens ATCC 13124; 6, human whole blood; 7, human leukocytes;
8, human liver; 9, Candida albicans ATCC 10231; and 10, H2O contamination control.
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Fragment analysis information.
The PCR products of all species
tested were initially visualized by UV illumination on ethidium
bromide-stained agarose gel to confirm successful amplification and
crude variability in length of the final product. The yeast family
demonstrated the highest level of interspecies variability (Fig.
3) compared to other fungi (data not
shown); however, size determination based on agarose gel
electrophoresis is not precise enough to unmistakably confirm species
identification.
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FIG. 3.
Detection of fungal species by agarose gel
electrophoresis. PCR amplification of variable Candida
species from culture colonies using the ITS4 and ITS86 universal fungal
primers was performed as described in Materials and Methods. Lanes: L,
123-bp ladder; 1, Candida albicans; 2, Candida
kefyr; 3, Candida zeylanoides; 4, Candida
tropicalis; 5, Candida krusei; 6, Candida
glabrata; 7, Candida guilliermondii; 8, Candida
lusitaniae; 9, Candida parapsilosis.
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To determine the degree of accuracy of the fragment analysis, multiple
strains of
Candida species were analyzed in multiple
runs on
the ABI PRISM 310 genetic analyzer, and the standard deviation
was then
calculated. The run-to-run variation resulted in a standard
deviation
0.5 bp (Table
1). Although the
variation between different
strains of a species was not extensively
tested, it was comparable
to the run-to-run variation of a single
strain, indicating that
the different strains tested were of the same
ITS2 length. The
documented instrument resolution provided by the
manufacturer
indicated a 1-base detection to 250 bp and a 2-base
detection
from 251 to 350 bp.
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TABLE 1.
Base pair determination and associated standard deviation
of the ITS2-containing PCR fragments of various species during
multiple fragment analysis runs
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The length of the ITS2 region amplicons was determined for 12
Candida species, 9 non-
Candida yeasts, 14 dermatophytes, and
21 other opportunistic and dematiaceous fungi (Table
2). Reference
strains were used for the
majority of the specimens, as well as
some clinical strains to test for
intraspecies consistency. Examples
of the electropherograms produced
from the GeneScan analysis are
shown in Fig.
4. Within 21 species of the yeast group,
18 ITS2
fragment lengths were determined.
Candida
stellatoides had the
same fragment length as
Candida
albicans (279 bp).
Candida zeylanoides,
Cryptococcus neoformans, and
Candida pelliculosa
(
H. anomala)
differed by only 1 bp (316, 315, and 317 bp
respectively).
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FIG. 4.
Electropherograms of five Candida species as
analyzed by the ABI PRISM 310 genetic analyzer. Control strains (graphs
1 to 5) were amplified by using ITS4 and fluorescently labeled ITS86
and dUTP as described in Materials and Methods. Each was run separately
on the capillary electrophoresis system along with an internal size
standard (GeneScan ROX-500). Standard peaks are shown as a separate
electropherogram (graph 6) for clarity of illustration. The standard
peak sizes are 139, 150, 150, 200, 240, 300, 340, 350, and 400 bp.
Graphs: 1, Candida albicans ATCC 10231; 2, Candida
tropicalis ATCC 66029; 3, Candida glabrata ATCC 90030;
4, Candida lusitaniae ATCC 42720; 5, Candida
krusei ATCC 6258.
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With the exception of
Epidermophyton floccosum (366 bp), all
of the dermatophytes were situated in a range between 298 and
325 bp,
with
Microsporum spp. in the higher range (313 to 325
bp)
and the
Trichophyton spp. in the lower range (298 to 315 bp).
Trichophyton rubrum and
Trichophyton
verrucosum had the same fragment
length, whereas the other
dermatophytes tested had a unique ITS2
length.
In addition to the yeasts and dermatophytes, 21 other medically
important fungi were tested and included opportunistic and
pathogenic
fungi. The four
Aspergillus species tested had close
fragment length sizes:
Aspergillus fumigatus, 284 bp;
Aspergillus flavus, 288 bp;
Aspergillus niger,
290 bp; and
Aspergillus terreus,
292 bp. Other
opportunistic fungi tested included
Fusarium solani (286 bp),
Absidia corymbifera (410 bp), and
Cunninghamella
bertholletiae (347 bp). The majority of the other molds had
unique ITS2 lengths;
however, a few fungi demonstrated fragments of
equal length or
nearly equal length. These included
Scedosporum
prolificans and
Cladophialophora carrionii, which were
both 303 bp;
Penicillium marneffei, which had the same
length as
Candida albicans; and
Exophiala
jeanselmei and
Sporothrix schenckii, which differed
by
1
bp.
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DISCUSSION |
The use of ribosomal DNA (rDNA) genes for identification of fungal
species is based on the detection of conserved sequences in 5.8S rDNA
and 28S rDNA that enable the amplification of the ITS2 region between
these two genes. In this study, we PCR amplified, by using a
fluorescent primer pair, the ITS2 regions of 56 fungal species of
clinical significance. The amplicons were rapidly and accurately sized
with an automated capillary electrophoresis system, ABI PRISM 310 genetic analyzer. No intraspecies variability was observed among
species for which more than one strain was tested. These species
included Candida albicans, Candida
guilliermondii, Candida tropicalis, Candida
krusei, Candida glabrata, Candida parapsilosis, Rhodotorula rubra, and Trichophyton
verrucosum. An exception was Trichosporon beigelii, of
which a reference strain and a clinical strain were tested. Kemker et
al. (12) previously demonstrated, by using restriction
fragment length polymorphism of a segment of the ribosomal genes
including the ITS2 region, that isolates identified as
Trichosporon beigelii are genetically distinct from each
other, depending on the source of the organism. The authors suggest
that Trichosporon beigelii may represent several distinct
entities and that, for the purpose of molecular diagnosis research,
isolates from invasive disease must be used as opposed to those from
culture collections.
The length of the amplicon containing the ITS2 region that has been
determined for each control organism may serve as reference for
specific fungal identification from clinical specimens, such as blood
and fresh tissue and paraffin-embedded specimens. Specific identification may not always be possible, since even with the precision of capillary electrophoresis, some ITS2 lengths are very
similar. However, even with a short list of causative organisms, combined with the clinical setting, site of infection, and histologic information, a presumptive identification can often be made. Of those
species likely to be isolated in blood specimens (Candida albicans, Candida tropicalis, Candida
glabrata, Candida parapsilosis, Candida
krusei, Candida lusitaniae, and Candida
pseudotropicalis [Candida kefyr]), all have a unique
ITS2 amplicon length. This may have implications for selection of
antifungal therapy. While fluconazole has become a drug of choice due
to its low level of toxicity, some non-albicans Candida
species, particularly Candida krusei, Candida
glabrata, and Candida parapsilosis (17, 21), have demonstrated fluconazole resistance, and another agent, such as
amphotericin B, should be chosen for empirical therapy. The ITS2 length
similarity of Candida stellatoidea and Candida
albicans is not surprising, since the genetically distinct
Candida stellatoidea type II has been classified as a
sucrose-negative variant of Candida albicans
(13), and this species is normally reported as Candida albicans. It is important to keep in mind the clinical importance of the organisms that need to be identified. The fact that the dermatophytes have very similar ITS2 lengths is not as great a concern
as, for example, the differentiation of dematiaceous from opportunistic
organisms. Thus, the ITS2 length in association with the clinical
picture can be an effective method for early diagnosis of fungal infection.
We are in the process of applying this method to clinical specimens. In
cases in which the ITS2 lengths may be similar, it is highly probable
that their sequences differ and may potentially be differentiated by
single-stranded conformation polymorphism (SSCP). This can also be
performed on a capillary electrophoresis system in the same time frame.
However, SSCP may detect intraspecies variations in the sequence and
therefore limit the specificity of the test. Another possibility may be
to include the amplification of the ITS1 fragment in a multiplex
PCR reaction, although this also increases the chance of detecting
intraspecies length differences if they occur. Further work is
necessary in a clinical setting to determine the specificity at which
our method will provide optimal clinically significant data.
The greatest impact this method may have in the clinical laboratory is
upon the time saved from sample collection to a final diagnosis. The
time to identification from specimen collection can be as little as
6.5 h, including the DNA extraction, amplification, and processing
of the first sample by the capillary electrophoresis. While at present,
the ABI PRISM 310 genetic analyzer is a single-capillary system which
runs one sample at a time (~30 min) for analysis, we anticipate the
availability of a multicapillary system in the near future which can
process multiple samples concurrently. Conventional blood cultures, on
the other hand, take an average of 3 to 7 days before positive fungal
identification. In addition, this technique is independent of organism
viability, growth, and biochemical and morphological phenotypes. This
technique requires only minimal technological expertise, in contrast to
conventional morphological methods. However, this method does not
establish antifungal susceptibilities, which must still be determined
by conventional methodology requiring growth of the organism. In
summary, we have developed a method for the comprehensive
identification of clinically relevant fungal isolates, which is
sensitive, rapid, and specific for most organisms tested.
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ACKNOWLEDGMENTS |
We thank Assunta Rendina and Evelyn Witwicki for their technical
assistance and expertise.
This project was supported in part by the Children's Hospital Research Foundation.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Microbiology, Health Sciences Centre, MS6-820 Sherbrook
Street, Winnipeg, Manitoba R3A 1R9, Canada. Phone: (204) 787-4696. Fax: (204) 787-4699. E-mail: cturenne{at}hc-sc.gc.ca.
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Abstract
Introduction
