Research Center for Pathogenic Fungi and
Microbial Toxicoses, Chiba University, Chuo-ku, Chiba (260-8673),
Japan,1 and
Faculty of Medical
Science, State University of Campinas, Campinas,
Brazil2
Received 3 August 1998/Returned for modification 10 September
1998/Accepted 15 October 1998
 |
INTRODUCTION |
Cryptococcus neoformans
is a cosmopolitan fungus and causes cryptococcosis in humans and
animals (7, 8). Recent increases in the numbers of AIDS
patients in Brazil have also led to increases in the numbers of
infections caused by this pathogenic fungus (5).
On the basis of the antigenic composition of its polysaccharide capsule
and biological differences, C. neoformans has been subdivided into two varieties and five serotypes, namely, C. neoformans var. gattii (serotypes B and C) and
C. neoformans var. neoformans (serotypes A,
D, and AD). It has been reported that the isolates of C. neoformans that infect AIDS patients are predominantly of serotype
A (7-9), and we also confirmed these findings of the predominance of serotype A by epidemiological studies with AIDS patients in Thailand (14). Isolation of a serotype D strain from AIDS patients has also been reported in France, Germany, and the
United States (3).
Several typing approaches have been used in epidemiological studies,
including serotyping and PCR fingerprinting (2, 6, 11).
Among them, the serotyping schemes have been found to be important
since the association of infection with serotype A strains in AIDS
patients was confirmed by recent epidemiological and ecological studies
(4, 10, 20). Random amplified polymorphic DNA (RAPD) fingerprinting has been demonstrated to have the ability to
discriminate between closely related isolates within a given population
(17, 18, 21). Ruma et al. (15) reported that RAPD
fingerprinting techniques are useful for distinguishing among the
varieties and serotypes of C. neoformans, and Varma and
Kwong-Chung (19) reported on similar results obtained with
various RAPD primers. No useful PCR primers derived from published RAPD
analyses of C. neoformans serotypes have been reported.
In the study described here, we developed new, useful PCR primer pairs
which should be specific for C. neoformans serotype A
or B on the basis of RAPD fingerprint pattern analyses of C. neoformans. The usefulness of other C. neoformans
serotype-specific PCR primer pairs, such as primer pairs specific for
serotypes C, D, and AD, for the identification of C. neoformans strains is also discussed.
| |
MATERIALS AND METHODS |
Microorganisms and determination of serotypes of C. neoformans strains.
The following isolates of C. neoformans were used as reference strains: for serotype A, strain
CDC 551; for serotype B, strain NIH 112; for serotype C, strain NIH 18;
for serotype D, strain IFM 5857; and for serotype AD, strain LY 23 (an
isolate from China). Thirty-three clinical C. neoformans isolates from the State University of Campinas were
used. The fungal strains were inoculated onto potato dextrose agar
(Difco) slants and were incubated at 30°C for approximately 48 to
72 h. After two transfers, the serotype of each strain was
determined by slide agglutination tests (Crypto Check; Iatron Co.,
Tokyo, Japan), and the serotypes were compared with those of the
reference strains of C. neoformans (14).
Extraction of DNA.
The chromosomal DNAs of all C. neoformans strains were extracted by a method modified from those
of Zhu et al. (22) and Poonwan et al. (14). The
strains were grown on brain heart infusion agar at 37°C for 1 week.
Three or four loopfuls of the fungal yeast cells from the agar slants
were suspended in 500 µl of TE buffer (100 mM Tris-HCl [pH 8.0], 1 mM EDTA) in an Eppendorf tube (1.5 ml) and then kept in a heater at
80°C for 30 min to kill the fungus. After shaking, the suspension was
centrifuged at 11,000 × g and the sediment was mixed with
0.5 ml of extraction buffer (100 mM Tris-HCl [pH 9.0] with 40 mM
EDTA), 0.1 ml of 10% sodium dodecyl sulfate, and 0.3 ml of benzyl
chloride. After shaking with a vortex mixer, the fungal cells were
incubated at 60°C for 30 min and then broken with a bead beater
(Wakenyaku Co., Kyoto, Japan) for 20 s. After centrifugation at
11,000 × g at 4°C for 5 min, the supernatant was
transferred to another Eppendorf tube, 3 M sodium acetate was added at
1/10 volume of the aqueous supernatant, and the mixture was cooled at
0°C for 10 min. The DNA was precipitated with 0.5 ml of 2-propanol
(
70°C, 3 h), and then the precipitate was washed with 70%
ethanol, dried, and resuspended in 100 µl of TE buffer. The DNA
concentration was determined with a UV monitor (Bio-mini UV monitor;
ATTO, Tokyo, Japan).
RAPD PCR analyses.
The following three primers were used for
the RAPD analysis: R-1 (5'-ATGGATCGGC-3'), R-2
(5'-ATTGCGTCCA-3'), and R-3 (5'-TCACGATGCA-3'). These were prepared on the basis of the reports of Goodwin and Annis (6) or on the basis of information from our
preliminary experiments. Amplification reactions were performed in a
volume of 30 µl of distilled water containing 2.5 µl of primer (20 pM), 2.5 µl of genomic DNA (1 µg/ml), and one PCR bead (Ready-to-Go PCR bead; Pharmacia Biotech). The PCR was performed by initially heating the samples at 94°C for 4 min; this was followed by 35 cycles
of denaturation at 94°C for 2 min, annealing at 32°C for 2 min,
extension at 72°C for 2 min, and a final extension at 72°C for 10 min in a thermoreactor. All reaction products were characterized by
electrophoresis on 1.5% agarose gels in 1× TBE (Tris-borate-EDTA) buffer at 80 V for 90 min and then stained in a solution of ethidium bromide at 0.5 µg/ml.
Extraction and sequencing of DNA from RAPD fingerprint
bands.
For extraction and sequencing of DNA from the RAPD
fingerprint bands, a RAPD PCR product was subjected to
phenol-chloroform extraction and was precipitated with ethanol. The DNA
fragments were cloned into the pGEM 3Zf vector and were transformed
into bacterial strain JM109 (Technical Manual; Promega Co., Madison, Wis.). Sequencing-grade plasmid DNA was purified with a plasmid purification kit (GFX Micro Plasmid Prep Kit; Pharmacia Biotech). The
sequencing was performed on an ABI prism 377A DNA sequencer using the
Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems
Division, Perkin-Elmer Japan Co. Ltd., Tokyo, Japan) according to the
manufacturer's protocol.
Determination of serotypes by PCR with a new PCR primer
pair.
The normal PCR with the newly prepared primer sets was
performed by initially heating the samples at 94°C for 10 min; this was followed by 25 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final 10-min extension step at 72°C. All the reaction products were analyzed by the same methods described above for
analysis of the RAPD fingerprint patterns.
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RESULTS |
Serotyping and RAPD analyses of clinical C. neoformans isolates in Brazil.
During 1996 and early 1998, we isolated 33 strains of C. neoformans from patients
in the hospital of the State University of Campinas, Campinas, Brazil.
Each isolate was classified into one of two serotypes, and there were
31 and 2 strains of serotypes A and B, respectively. No serotype C, D,
or AD strain was observed.
The RAPD fingerprint patterns of 31 strains of C. neoformans serotype A obtained with three different 10-mer primers
were analyzed. The fingerprint patterns of 31 strains of C. neoformans serotype A isolated in Brazil were shown to be similar
to each other when we used the three 10-mer PCR primers. The RAPD
patterns for seven representative strains obtained with the R-1 primer are shown in Fig. 1. We compared these
fingerprint patterns for the Brazilian isolates with those for
reference serotype A strains and found that they were also similar to
each other and that most of the bands were common for each serotype A
strain tested, although some minor variations were observed (data not
shown).
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FIG. 1.
RAPD fingerprint patterns of seven strains of
C. neoformans serotype A isolated in Brazil obtained
with primer R-1. Lane M, DNA ladder marker used as the molecular size
standard; lanes 1 to 7, clinical serotype A isolates from Brazil.
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When we used three different 10-mer primers for RAPD amplification of
C. neoformans serotype B, each primer also showed a characteristic RAPD fingerprint pattern (data not shown).
Therefore, we were interested in the RAPD patterns of the reference
C. neoformans serotype A, B, C, D, and AD strains. When we compared the patterns of these reference strains obtained with the
three 10-mer primers, we found that the pattern obtained with each
primer was serotype specific. Among the three primers, since primer R-2
was found to produce on the electrophoresis gel PCR bands that were
sharper than those produced by the other two primers, it was selected
for use in the following studies for sequence determination. The RAPD
patterns of the reference serotype A, B, C, D, and AD strains obtained
with primer R-2 are shown in Fig. 2, and
the DNA bands that we used to determine the sequence are indicated by
arrows.
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FIG. 2.
RAPD fingerprint patterns of the reference C. neoformans serotype A, B, C, D and AD strains obtained with primer
R-2. Lane M, marker; lanes A to AD, reference serotype A, B, C, D, and
AD strains, respectively. The reference strains were CDC 551 (serotype
A), NIH 112 (serotype B), NIH 18 (serotype C), IFM 5857 (serotype D),
and LY 23 (serotype AD). The arrows indicate the DNA bands that were
obtained by RAPD analysis and that were extracted and sequenced in the
present experiment.
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Sequence determination and preparation of new PCR primers.
For
the preparation of serotype-specific PCR primers for the detection of
C. neoformans serotypes A and B, bands of about 700 and
450 bp, respectively, were extracted. After purification of the DNA
fragments, they were cloned into the pGEM 3Zf vector, and the sequences
were determined with an automated DNA sequencer as described in
Materials and Methods. The DNA sequences of the extracted bands are
presented in Fig. 3.
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FIG. 3.
Newly determined sequence of 695-bp band of DNA from a
C. neoformans serotype A strain obtained by RAPD
analysis and the sequences (underlined) of the new PCR primer pair that
was prepared. Newly determined sequence of 448-bp band of DNA from a
C. neoformans serotype B strain obtained by RAPD
analysis and the sequences (underlined) of the new PCR primer pair that
was prepared.
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Preparation of new PCR primers for detection of serotype A.
The sizes of the DNA bands for the detection of serotypes A and B were
695 and 448 bp, respectively (Fig. 3A and B). On the basis of the DNA
sequence information, two PCR primer pairs believed to be specific for
C. neoformans serotype A and to amplify the 695-bp
fragment (the CNa-70-Sand CNa-70-A primer pair) and the 579-bp fragment
(the CNa-29-S and CNa-29-A primer pair) were prepared (Tables
1 and 2).
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TABLE 1.
PCR primers prepared on the basis of the DNA
sequence information for the RAPD fingerprint pattern bands
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As shown in Fig. 4, PCR with the CNa-70-S
and CNa-70-A primer pair amplified a 695-bp DNA fragment from the
reference C. neoformans serotype A strain (CDC 551) and
three clinical strains of serotype A isolates. The primer pair did not
amplify the DNA band from the reference C. neoformans
serotype B (NIH 112), C (NIH 18), D (IFM 5857), or AD (LY 23) strain
when three reference strains were used. When we also tested 31 strains
of C. neoformans serotype A clinical isolates with this
primer pair (CNa-70-S and CNa-70-A), the 695-bp DNA fragment was
amplified from all strains (data not shown).
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FIG. 4.
PCR identification of C. neoformans
serotype A strains with the CNa-70-S and CNa-70-A PCR primer pair. Lane
M, marker; lanes A to AD, reference strains of serotypes A, B, C, D,
and AD, respectively; lanes CA, clinical serotype A isolates (CAMP-1,
CAMP-2, and CAMP-4 from left to right, respectively) from Brazil.
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We also tested a PCR primer pair (the CNa-29-S and CNa-29-A primer
pair) which was designed to amplify the internal sequence within the
695-bp band. This primer pair amplified a 579-bp fragment of DNA from
C. neoformans serotype A strains, including 31 of the
clinical isolates and the reference strains. There was no amplification
of DNA from the reference serotype B, C, D or AD strain (data not shown).
As shown in Fig. 5, PCR with the CNa-29-S
and CNa-70-A primer pair amplified a 666-bp fragment of DNA from
C. neoformans serotype A along with another amplified
satellite 460-bp band. When we tested 32 clinical isolates, the primer
pair amplified DNA from all C. neoformans serotype A
strains, which showed 666-bp bands along with the 460-bp satellite
band. This PCR primer pair was also found to amplify DNA from
C. neoformans serotype B and C reference strains, which
showed a PCR product of about 290 bp.
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FIG. 5.
PCR identification of C. neoformans
serotype B strains with the serotype A-specific CNa-29-S and CNa-70-A
PCR primer pair. Serotype A and B strains were identified by the
presence of amplified bands of 666 bp (along with one of 460 bp) and
290 bp, respectively. Lane M, marker; lanes A and B, reference strains
of serotypes A (CDC 551) and B (NIH 112), respectively; lanes CA,
clinical serotype A isolates (CAMP-1, CAMP-2, and CAMP-4 from left to
right, respectively) from Brazil.
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Preparation of new PCR primers for serotype B.
The CNb-49-S
and CNb-49-A PCR primer pair (Tables 1 and 2) amplified DNAs from
clinical C. neoformans serotype B isolates from Brazil
(strains CAMP-3 and CAMP-24) and the reference serotype B strains (NIH
112), and amplification of DNA from a C. neoformans serotype C strain (strain NIH 18) was also observed (data not shown).
The PCR identification of two clinical strains of C. neoformans serotype B is shown in Fig.
6. This PCR primer combination was not
able to amplify DNA from reference C. neoformans
serotype A, D, or AD strains.
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FIG. 6.
PCR identification of two clinical strains of
C. neoformans serotype B with the CNb-49-S and CNb-49-A
PCR primer pair. Lane M, marker; lanes A and B, reference strains of
serotypes A (CDC 551) and B (NIH 112), respectively; lanes CB, serotype
B clinical isolates (CAMP-3 and CAMP-24 on the left and right,
respectively) from Brazil.
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DISCUSSION |
It has been reported that C. neoformans serotype A
is distributed worldwide and is a major cause of all clinical types of cryptococcosis (7). C. neoformans serotype
B, on the other hand, has a more restricted global distribution and is
found in subtropical to tropical countries. Since Brazil is located in a tropical region, the existence of cryptococcosis due to serotype B is
likely. Kwon-Chung et al. (9) reported that C. neoformans var. neoformans constituted about 98 to
100% of isolates from AIDS patients in Brazil and Southern California
and that 40 to 45% of the isolates from non-AIDS patients were
serotype B. Therefore, the present data indicating that 31 of 33 strains were C. neoformans serotype A may be
reasonable, because most of the present C. neoformans strains from Brazil were isolated from AIDS patients.
Molecular techniques such as restriction fragment length polymorphism
analysis and RAPD analysis have provided powerful tools for the direct
analysis of the genomes and for epidemiological studies of many
pathogens (1, 10, 16, 21). Our data showed that the RAPD
fingerprint patterns of 31 clinical isolates of C. neoformans serotype A are very similar, suggesting that most of
the Brazilian C. neoformans serotype A isolates were
genetically homogeneous, although minor variations were obtained by
RAPD analysis with primer R-1 (Fig. 1). The reason for the genetic
homogeneity of these Brazilian isolates of C. neoformans serotype A by RAPD analyses is not clear, but it may be
due to the limited region from which strains were isolated, that is,
the State University of Campinas. To confirm this homogeneity of
Brazilian isolates of C. neoformans serotype A,
comparative RAPD studies with different primers and other strains from
different regions of Brazil are in progress at the State University of
Campinas, Campinas, Brazil.
Ruma et al. (15) reported that RAPD analysis techniques can
distinguish between serotype A, D, or AD strains of C. neoformans var. neoformans. They also reported that
separation of serotype B or C C. neoformans var.
gattii strains by the RAPD method is possible. Varma et al.
(20) analyzed 156 strains of C. neoformans according to their RAPD fingerprint patterns and concluded that C. neoformans var. neoformans and
C. neoformans var. gattii can be classified
into several groups according to their distinctive DNA fingerprint
patterns. Our present RAPD studies also indicated that discrimination
of serotypes according to these patterns is plausible.
Although many reports have shown that RAPD pattern analysis is useful
for the identification of the serotypes of C. neoformans, developmental studies of a C. neoformans serotype-specific primer pair that could be useful for
PCR have not been documented. In the experiments described here, we
prepared PCR primer pairs which were specific for serotype A or B
strains because these were the only two serotypes isolated from the
samples from Brazil. On the basis of the information obtained from RAPD
analysis, we designed and prepared two and one PCR primer pairs which
were expected to be specific for serotypes A and B, respectively. Use
of these pairs (CNa-70-A and CNa-70-A, CNa-29-S and CNa-29-A, and
CNb-49-S and CNb-49-A) for the identification of serotypes was 100%
successful when we tested samples of DNA from 31 strains of
C. neoformans serotype A and 2 strains of C. neoformans serotype B, including each reference strain. To our
knowledge, this is the first report of primers that can be used for the
serotype determination (serotype A or B) of C. neoformans strains by PCR.
Furthermore, among four PCR primers specific for serotype A, a
different primer pair (CNa-29-S and CNa-70-A) will be useful for the
rapid detection of the two serotypes of fungal pathogens (serotypes A
and B) in Brazil. Discrimination of the two serotypes by previously
used routine biological methods has not been easy in clinical
laboratories in Brazil which handle samples from AIDS patients, and
C. neoformans serotype A and B strains are major clinical isolates at the hospital of the State University of Campinas. The advantage of using this PCR primer pair is that an experiment in
one PCR tube can simultaneously identify serotype B and C strains as
well as serotype A strains. It is not clear why the serotype A-specific
PCR primer pair (CNa-29-S and CNa-70-A) is able to amplify DNA (about
290 bp) from serotype B or C strains. Since the sequence information
for the 290-bp bands is expected to give us useful information,
analysis of the sequences is in progress.
Although two separate RAPD fingerprint patterns were recognizable for
serotype B and C strains, our present PCR primer pair could not
discriminate between the two serotypes. This was a preliminary experiment, but we were successful in preparing two other PCR primer
pairs which are expected to be specific for serotype C. These two
serotype C-specific PCR primer pairs also amplified DNAs from serotype
B strains, however. These data may have some correlation with those in
the report of Ruma et al. (15) since they were successful in
differentiating serotype B and C strains only with one of seven primers
tested, although the differentiation of strains of other serotypes was
easier when the strains were tested by RAPD fingerprint pattern
analyses. These data are therefore believed to suggest that serotypes B
and C are genetically homogeneous.
During the specificity studies, we also found that the CNa-70-S and
CNa-70-A PCR primer pair amplified DNA (about 500 bp) from
Cryptococcus albidus, in addition to C. neoformans serotype A. Although more detailed studies are needed,
this primer pair should be a promising and useful candidate for the
identification of the C. albidus fungus, which is
sometimes isolated from clinical specimens.
Mitchell et al. (12) reported on a general PCR primer pair
(CN4 and CN5) which can identify all varieties of C. neoformans, and we confirmed the usefulness of this primer pair
for the identification of all serotypes of C. neoformans. Although our present PCR primer pairs cannot identify
serotype D and AD strains, use of their PCR primer pair combined with
the present serotype-specific PCR primer pair may allow the complete
differentiation of C. neoformans strains into either
C. neoformans var. neoformans or
C. neoformans var. gattii.
Throughout preliminary experiments, we were able to prepare a new PCR
primer pair (CNad-4-sense [5'-ATTGCGTCCAGATAACGATTC-3'] and CNad-4-antisense [5'-ATTGCGTCCACACCGCGTGGA-3']
for the detection of serotype AD strains and a primer pair
(CNd-2-sense [5'-ATTGCGTCCAGCATACGAATT-3'] and
CNd-2-antisense [5'-ATTGCGTCCACATCATGCAGC-3']) for the
detection of serotype D strains on the basis of the band sequence
information obtained for each reference serotype strain by RAPD
analysis. Each PCR primer pair for the detection of serotypes AD and D
was designed to amplify bands of 417 and 244 bp, respectively. Our preliminary experimental results showed that the primer pair specific for serotype AD amplified DNA (417 bp) from C. neoformans serotype A, AD, and D reference strains and was not
specific for serotype AD. On the other hand, the PCR primer pair for
serotype D amplified DNA (244-bp) from the reference serotype D strain
but did not amplify DNA from serotype A or AD strains. Interestingly,
the PCR primer pair amplified DNA from serotype C and B strains. Since we have only a few C. neoformans serotypes AD and D
strains in our laboratory, we were unable to clearly determine the
specificities of the PCR primer pairs. Detailed studies of this with
other several serotype AD and D strains are planned.
The sequences of the 695- and 448-bp fragments seem to be related to
certain bacterial and fungal aminotransferases and mammalian and fungal
DNA polymerases (as determined with the FAST program) (13).
The relation of the supposed serotype specificity of the PCR primer
pairs to antigenic differences among capsular polysaccharides of
C. neoformans is not clear. However, since the
subdivision of C. neoformans into varieties is not
based solely on antigenic determinants of the polysaccharide capsule
but is based on biological differences (8, 9, 17), further
detailed genetic and biochemical studies are necessary to elucidate the
relation of the specific PCR primer and antigenic differences.
Financial support was provided by JICA (Japan International
Cooperation Agency) under the project The Clinical Research Project in
the State University of Campinas in Brazil.
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Abstract
Introduction
