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Journal of Clinical Microbiology, October 2001, p. 3466-3471, Vol. 39, No. 10
Institute of Biomedical
Engineering1 and Department of Medical
Technology, College of Medicine,4 National Cheng
Kung University, Department of Pathology, National Cheng Kung
University Hospital,2 and Department
of Clinical Pathology, Linko Medical Center, Chang Gung Memorial
Hospital,3 Tainan 701, Taiwan, Republic of
China
Received 6 December 2000/Returned for modification 30 April
2001/Accepted 20 July 2001
Yeasts are emerging as important etiological agents of nosocomial
bloodstream infections. A multiplex PCR method was developed to rapidly
identify clinically important yeasts that cause fungemia. The method
amplified the internal transcribed spacer 1 (ITS1) region between the
18S and 5.8S rRNA genes and a specific DNA fragment within the ITS2
region of Candida albicans. With this method, C. albicans produced two
amplicons, whereas other species produced only one. Through sequence
analysis, the precise lengths of the PCR products were found to
be as follows: C. glabrata (482 or 483 bp), C. guilliermondii (248 bp),
C. parapsilosis (229 bp), C. albicans (218 or 219 and 110 bp), C. tropicalis (218 bp), Cryptococcus neoformans (201 bp),
and C. krusei (182 bp). The PCR products could be effectively separated by disk polyacrylamide gel
electrophoresis. The method was used to test 249 positive blood
cultures (255 isolates), from which the following species (strain
number) were isolated: C. albicans (128),
C. tropicalis (51), C.
glabrata (28), C.
parapsilosis (23), C.
neoformans (9), C.
krusei (5), C.
guilliermondii (3), and other, minor species (8). The
test sensitivity of the method was 96.9% (247 of 255 isolates). The
eight minor species were either misidentified (one strain) or not
identified (seven strains). From the time at which a positive bottle
was found, the multiplex PCR could be completed within 8 h; the
present method is simpler than any previously reported molecular method
for the identification of blood yeasts.
Yeasts are emerging as important
etiological agents of bloodstream infections (22, 23), a
complication associated with a high mortality rate (1, 3).
This problem is compounded by an increase in resistance to antifungal
agents, particularly the azoles (8, 18, 22, 25, 28, 29,
31) and amphotericin B (19). Bloodstream fungal
infections constitute a serious health problem because of the excessive
hospital stay, added health care costs, and high morbidity and
mortality attributed to the diseases (39).
Candida albicans, C. tropicalis, C. glabrata, C. parapsilosis, C. krusei, and
Cryptococcus neoformans are the most common yeasts causing bloodstream infections (2, 23). These six species may account for 95 to 98% of all blood yeasts (19, 23, 27). C. guilliermondii and other, minor
species may be isolated occasionally (2, 32). The rates of
isolation of the major yeast species causing fungemia have been
determined in several studies: C. albicans (50 to
59%), C. tropicalis (11 to 25%), C. glabrata (8 to 18%), C. parapsilosis
(7 to 15%), C. krusei (2 to 4%), C. neoformans (~2%), and other species (~2%) (1,
19, 23, 27, 28). Therefore, rapid identification of blood yeasts could be targeted solely for these species, although the possibility of
other, rarely encountered species always exists.
Fluconazole, which has a low level of toxicity, has been reported to be
as effective as amphotericin B for the treatment of candidemia in
patients without neutropenia (26), although C. glabrata and C. krusei are innately
more resistant to fluconazole. The MIC50s of
fluconazole for C. krusei and C. glabrata are 32 and 16 µg/ml, respectively; both values
are much higher than those for C. albicans (0.25 µg/ml), C. tropicalis (1 µg/ml), and
C. parapsilosis (1 µg/ml) (23).
Therefore, earlier information regarding the species causing fungemia
may help physicians to select appropriate antifungal agents and
regimens to treat patients. The rate of isolation of C. glabrata from blood cultures has increased from 8% during
the period from 1952 to 1992 to 18 to 20% in recent surveys (22,
23). This increase might be due to the widespread use of
fluconazole for prophylaxis and treatment of candidiasis, affirming the
need for more rapid and accurate identification.
At present, the identification of yeasts in positive blood cultures by
use of conventional morphological and metabolic characteristics requires from one to several days after isolation. In order to decrease
that time, methods devised for the rapid diagnosis of fungal infections
include detection of antibody (42), cell wall mannan
(5), enolase (37), and specific antibody in
combination with PCR to detect C. albicans DNA
(15). Efforts have been directed toward molecular
testing, such as the use of rRNA genes (rDNA), for species
identification. PCR followed by hybridization of the amplicons with
species-specific probes has also been used to detect a variety of fungi
(6, 7, 9-11, 21, 24, 30, 35, 36). Nested PCR (13,
17, 20, 33) or PCR followed by restriction enzyme analysis
(16, 41) has also been used to detect several fungal
pathogens. The above methods have shown promise for the diagnosis of
fungal infections but have problems that prevent their routine use in a
clinical laboratory. For example, the DNA hybridization technique
normally involves multiple steps of incubation and washing under
stringently controlled conditions, which are both time-consuming and
cumbersome. The use of nested PCR or PCR in conjunction with
restriction enzyme analysis, however, may add needless complexity to
assay procedures.
Recently, a fluorescent capillary electrophoresis system was developed
to identify fungi by use of the length variability of the internal
transcribed spacer 2 (ITS2) genetic region (34). However,
the fragment lengths of the ITS2 regions were similar in several
important yeasts that cause fungemia, thereby preventing the
identification of some species. The aim of the present study was to
evaluate a multiplex PCR method for the identification of
C. neoformans and Candida species
that are frequently isolated from blood cultures. The method was based
on the size variability of the ITS1 regions in different species and on
the amplification of a specific DNA fragment of the ITS2 region of
C. albicans.
Yeast strains.
A total of 22 stock yeast cultures were used
in this study (Table 1). Among these
cultures, 18 strains were obtained from the Culture Collection and
Research Center (CCRC, Hsinchu, Taiwan), and the remaining 4 strains
were clinical isolates.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3466-3471.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Rapid Identification of Yeasts in Positive Blood
Cultures by a Multiplex PCR Method
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Pure yeast cultures used in this study and the
lengths of PCR products
DNA extraction from pure cultures.
Stock cultures of yeasts
were subcultured on Sabouraud dextrose agar (Difco, Detroit, Mich.) and
incubated at 37°C. Colonies of these strains were suspended in
saline to obtain the turbidity of a 0.5 McFarland standard at a 530-nm
wavelength. Two microliters of cell suspension was added to 18 µl of
microLYSIS solution (Microzone Limited, East Sussex, United Kingdom) in
a 0.2-ml Eppendorf tube and overlaid with 20 µl of sterilized mineral
oil. The tube was heated in a thermal cycler (OmniGen; Hybaid Limited,
Middlesex, United Kingdom) using the following temperature profile, as
recommended by the manufacturer: 65°C, 5 min; 96°C, 2 min; 65°C,
4 min; 96°C, 1 min; 65°C, 1 min; 96°C, 30 s; and 30°C, 5 min. After cycling, the lysis solution-DNA mixture was used directly
for PCR amplification or stored at 
20°C for further use.
Escherichia coli ATCC 25922, Staphylococcus aureus 0400, Klebsiella
pneumoniae 03583, Enterobacter cloacae
00109, and Streptococcus pneumoniae 0424 were
cultivated on blood agar at 37°C for 18 to 24 h, and the
bacterial DNA was extracted in a manner similar to that used for pure
yeast cultures.
Clinical specimens. Blood samples were collected from the National Cheng Kung University Medical Center, Tainan, Taiwan, and from Chang Gung Memorial Hospital. BACTEC blood culture bottles (Becton Dickinson Microbiology Systems, Cockeysville, Md.) were normally inoculated with 3 to 10 ml of blood from patients, inserted into the BACTEC NR660 instrument (Becton Dickinson Microbiology Systems), and incubated at 37°C. Gram stain smears of aliquots from positive bottles were prepared to check for the presence of yeasts. A total of 249 positive blood culture bottles containing yeasts were analyzed in this study. The blood yeasts isolated on subculture plates were identified by conventional procedures based on phenotypic and biochemical reactions (38).
Isolation of yeast DNA from positive blood cultures. The method of Fujita et al. (9) was used with a small modification to extract yeast DNA from the positive culture broths. An aliquot (0.2 ml) of positive broth containing yeasts was added to 0.8 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) containing 0.05% proteinase K (Worthington Biochemical Inc., Lakewood, N.J.) and 0.05% Tween 20. The cell suspension was incubated at 55°C for 30 min and then centrifuged at 8,000 × g for 10 min in a microcentrifuge. The pellet was washed with 0.5 ml of TE buffer containing 0.5% Tween 20 and then with 0.5 ml of SE solution (1 M sorbitol, 0.1 M EDTA). After centrifugation at 8,000 × g for 10 min, the pellet was suspended in 0.5 ml of Lyticase solution (10 mg/ml; Sigma Chemical Co., St. Louis, Mo.) and incubated at 37°C for 1 h. After centrifugation, the pellet was suspended in 10 µl of TE buffer, and 1 µl of the suspension was added to 19 µl of microLYSIS solution. The suspension was heated in a thermal cycler to extract yeast DNA as previously described for pure cultures. Seven randomly selected positive blood cultures containing bacteria were processed in the same manner for DNA extraction. In addition, DNA was extracted from two blood samples from healthy individuals for PCR assay.
PCR amplification. The fungus-specific, universal primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS2 (5'-GCT GCG TTC TTC ATC GAT GC-3') (40) were used to amplify a small conserved portion of the 18S rDNA region, the adjacent ITS1, and a small portion of the 28S rDNA region. In addition, C. albicans-specific primers CA3 (5'-GGT TTG CTT GAA AGA CGG TAG-3') and CA4 (5'-AGT TTG AAG ATA TAC GTG GTA G-3') (12) were also included in the PCR mixture to amplify a portion of the ITS2 region of C. albicans. The four primers (ITS1, ITS2, CA3, and CA4) were synthesized at TIB MOLBIOL (Berlin, Germany). Multiplex PCR was performed with 2 µl (1 to 5 ng) of template DNA in a total reaction volume of 50 µl consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 3.2 µM primers (ITS1 and ITS2, 0.4 µM each; CA3 and CA4, 1.2 µM each), Taq DNA polymerase (1.25 U), and 50 µl of a mineral oil overlay. PCR was carried out with an OmniGen thermal cycler under the following conditions: initial denaturation, 94°C, 3 min; 35 cycles of denaturation (94°C, 1 min), annealing (60°C, 1 min), and extension (72°C, 1 min); and final extension, 72°C, 5 min. A negative control run was performed with each test run by replacing the template DNA with sterilized water in the PCR mixture. A positive culture broth containing C. albicans was run in parallel with unknown samples, and this culture broth was used as a positive control.
Limit of detection of C. albicans in blood. To determine the limit of detection of the multiplex PCR, whole blood was seeded with C. albicans CCRC 20512 to reach a concentration of 2 × 105 CFU/ml. The seeded blood was serially diluted 10-fold with whole blood, and 0.2 ml of the diluted samples was used for PCR as described above. The cell numbers (CFU per milliliter) of the diluted cell suspensions were determined by the plate count method (11) with Sabouraud dextrose agar as the culture medium. Plates were incubated at 35°C for 48 h before enumeration.
Determination of the lengths of the PCR products. To determine the precise lengths of the PCR products amplified by primers ITS1 and ITS2, the amplicons were purified by using a PCR cleanup kit (Viogene, Sunnyvale, Calif.) and were directly cycle sequenced in both directions with an ABI Prism 377 automated system (Applied Biosystems, Taipei, Taiwan). The size of the fragment amplified from C. albicans by primers CA3 and CA4 was determined in a similar way. For each species, two to five strains were sequenced in both directions to determine the precise lengths of the amplicons (Table 1). The PCR products of several minor species (C. famata, C. lusitaniae, C. pelliculosa, Rhodotorula rubra, and Trichosporon beigelii) isolated in this study were also sequenced to determine the precise lengths of their amplicons.
Disk PAGE. PCR products were analyzed by disk polyacrylamide gel electrophoresis (PAGE) (4) with a minigel system (Mini-Protean II; Bio-Rad, Hercules, Calif.). The running gel had an acrylamide concentration of 9% and was 0.75 mm in thickness. The time required for running electrophoresis was 3 h. After electrophoresis, the gels were stained with ethidium bromide (0.5 µg/ml) and viewed with an IS-1000 digital imaging system (Alpha Innotech Corporation, San Leandro, Calif.). In addition to the 50-bp DNA ladder, equal amounts (20 µl) of the PCR products amplified from C. glabrata CCRC 20586 (482 bp), C. guilliermondii CCRC 21500 (248 bp), C. parapsilosis CCRC 20515 (229 bp), C. albicans CCRC 20512 (219 and 110 bp), C. tropicalis CCRC 20520 (218 bp), C. neoformans CCRC 20528 (201 bp), and C. krusei CCRC 20514 (182 bp) were mixed to serve as markers for species identification. The species markers were run in parallel with the PCR products amplified from unknown samples to facilitate side-by-side comparisons between the markers and the PCR products amplified from the blood samples.
Definition of test sensitivity and specificity. For identification of the seven yeast species (C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, and C. neoformans) in blood cultures, the sensitivity of the multiplex PCR was defined as the number of strains of these species correctly identified (true positives) divided by the total number of yeast strains isolated. The test specificity was defined as the number of strains which did not belong to the above seven species and were not identified as any one of the seven major species (true negatives) divided by the total number of strains not included in these seven species (14).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Fragment analysis of PCR products.
Through sequence analysis,
the precise lengths of the PCR products amplified by the
fungus-specific, universal primers ITS1 and ITS2 were determined:
C. krusei (182 bp), C. neoformans (201 bp), C. tropicalis
(218 bp), C. albicans (218 or 219 bp),
C. parapsilosis (229 bp), C. guilliermondii (248 bp), and C. glabrata (482 or 483 bp) (Table 1). Different strains of the
same species produced PCR products having the same length or differing
in length by only 1 bp, and the PCR products of different species could
be separated more easily by disk PAGE than by agarose gel
electrophoresis. This was especially true for separating C. parapsilosis (229 bp) from C. tropicalis (218 bp). Another problem was that amplicons of
the ITS1 regions of C. albicans (218 or 219 bp)
and C. tropicalis (218 bp) had the same mobility
on polyacrylamide gels (Fig. 1, lanes 4 and 6). In order to discriminate between these two species, primers CA3
and CA4, which are specific for C. albicans
(12), were included in the PCR mixture, and an additional
product was obtained for the organism (Fig. 1, lane 4). With the
multiplex approach, C. albicans produced two
amplicons (218 or 219 and 110 bp) (Fig. 1, lane 4), whereas each of the
remaining six species produced only one.
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Limit of detection of the PCR. The limit of detection of the multiplex PCR for C. albicans CCRC 20512 artificially inoculated in whole blood was approximately 20 CFU/ml (data not shown). With serially diluted DNA in water, the limit of detection of the PCR was 4 pg of C. albicans DNA per assay. The limit of detection of yeast DNA was very close to that (10 pg DNA) reported by Jaeger et al. (13) and was approximately equal to 100 cells (37 fg of DNA per cell of C. albicans).
Identification of yeasts in positive blood cultures.
A total
of 249 positive blood cultures containing yeasts were analyzed by the
multiplex PCR for species identification. From these blood cultures,
255 strains of yeasts were isolated. The most frequently isolated
species was C. albicans (128 strains, 50.4%),
followed by C. tropicalis (51 strains, 19.7%),
C. glabrata (28 strains, 11%), C. parapsilosis (23 strains, 9.1%), C. neoformans (9 strains, 3.5%), C. krusei (5 strains, 2%), C. guilliermondii (3 strains, 1.2%), and other species (8 strains, 3.1%) (Table 2). All strains of
the above species, except for the eight minor species, were correctly
identified, resulting in a test sensitivity of 100% for each of the
above seven species (Table 2). However, the test sensitivity for the
PCR assay was 96.9%, based on the total number (247 strains) of yeasts
identified divided by the total number (255 strains) of yeasts
isolated.
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Specificity of the multiplex PCR.
The eight miscellaneous
strains included three strains of Candida spp. and one
strain of each of the following species: C. famata, C. lusitaniae, C. pelliculosa, R. rubra, and T. beigelii. As shown in Fig. 3, the
electrophoretic mobilities of amplicons of C. pelliculosa (262 bp, lane 2), C. famata (236 bp, lane 3), T. beigelii
(196 bp, lane 6), C. lusitaniae (147 bp, lane 8), and one undetermined Candida species (lane 9) were
different from those of the seven species markers; hence, the five
species were not identified. However, the PCR products amplified from
R. rubra (232 bp; Fig. 3, lane 5) and
C. parapsilosis (229 bp, lane 4) were only 3 bp
apart; therefore, R. rubra was misidentified as C. parapsilosis, resulting in a test specificity
of 87.5% (seven of eight strains). The relatively low specificity was
due to the limited proportion (3.1%; 8 of 255 strains) of minor yeast
species recovered from positive blood cultures.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This report describes the use of multiplex PCR to identify the most frequently encountered yeasts in blood cultures. The method used universal fungal primers ITS1 and ITS2 to amplify a conserved portion of the 18S rDNA region, the adjacent ITS1 region, and a small portion of the 5.8S rDNA region, yielding products with variable sizes among the major species causing fungemia. Another primer pair (CA3 and CA4) was used to amplify a specific DNA fragment of the ITS2 region of C. albicans. With disk PAGE, the PCR products could be effectively separated and recognized, even though they differed by a few base pairs. From the time at which a blood culture become positive, the multiplex method reduced the identification time for yeasts from approximately 1 to 3 days by routine identification methods to about 8 h. Another advantage of the method was that multiple yeast species coexisting in a blood culture could be detected at the same time (Fig. 2, lanes 2 to 4).
It is generally perceived that DNA extraction from yeasts either by lysis of enzymes (9, 21, 36) or by bead sonication (34) followed by phenol-chloroform extraction is the most tedious and cumbersome step of a PCR-based identification method and limits its use in a routine clinical laboratory. We found that a commercial extraction kit (microLYSIS) was an effective and simple method for extracting DNA from pure yeast cultures within 30 min. The only step used for DNA extraction with this kit was heating of yeast cell suspensions in the lysis solution in a thermal cycler, eliminating the use of phenol-chloroform and alcohol for DNA purification and precipitation, respectively. However, for extraction of yeast DNA from blood cultures, we found that a prior step of lysing blood cells with proteinase K followed by Lyticase digestion of the yeast cell walls was necessary to obtain good results.
The efficacy of the multiplex method relies on several factors. First,
the concentration of yeast cells in positive blood cultures normally
exceeds 105 CFU/ml (2). Second,
fungal rDNA has a high copy number (40 to 80 copies per haploid genome)
(40). Third, fungemia is usually caused by a limited
number of fungal species. The most commonly encountered yeast species
(C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, C. krusei, and
C. neoformans) may represent 
95% of the
total yeasts recovered from blood cultures (19, 23, 26).
Finally, Turenne et al. determined that universal fungal primers ITS3 and ITS4 and an automated system of fluorescent capillary electrophoresis could be used to determine the sizes of amplicons of the ITS2 genetic regions of some fungi (34). However, even with this sophisticated technique, it was still difficult to differentiate the PCR products of C. albicans (279 bp) and C. krusei (282 bp) by chromatographic retention times. Adapting this approach to the multiplex PCR method developed in this study, however, produced a method with a high sensitivity (96.9%). The relatively low specificity (87.5%) was due to a limited proportion (3.1%; 8 of 255 strains) of the minor yeast species recovered from our blood cultures. Starting from a positive blood culture, this method can be completed within 8 h and is simpler than any previously reported molecular method for the identification of blood yeasts.
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ACKNOWLEDGMENTS |
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This work was supported by grant NSC 89-2314-B-006-101 from the National Science Council, Taipei, Taiwan, Republic of China.
We thank the medical technicians at the Department of Pathology, National Cheng Kung University Medical Center, for help in identifying all the clinical blood isolates.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Technology, College of Medicine, National Cheng Kung University, 1 University Rd., Tainan 701, Taiwan, Republic of China. Phone: 886-6-235-3535, ext. 5790. Fax: 886-6-236-3956. E-mail: tsungcha{at}mail.ncku.edu.tw.
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Materials and Methods
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Discussion
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Journal of Clinical Microbiology, October 2001, p. 3466-3471, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3466-3471.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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