Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans, with Implications for Diagnosis
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微生物临床杂志 2006年第1期
Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
SAIC-Frederick, Inc., Frederick, Maryland
Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, Maryland
ABSTRACT
Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of C. albicans incubated in Hanks balanced salt solution (±10% bovine serum albumin [BSA]) or RPMI (±10% BSA) showed a significant release of DNA at T equal to 24 h compared to T equal to 0 h (P 0.01). C. albicans incubated with H-MNCs exhibited a greater release of DNA than C. albicans cells alone over 24 h (P = 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of C. albicans, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by C. albicans collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of C. albicans DNA.
INTRODUCTION
Invasive candidiasis remains one of the leading causes of morbidity and mortality in immunocompromised and critically ill patients (1, 8, 9, 15, 18, 20, 23), with Candida albicans infections comprising 45% of all Candida bloodstream infections (10). Since the earliest introduction of PCR for the detection of C. albicans in blood (3, 12), this technology has been increasingly refined for molecular detection of deeply invasive candidiasis. Real-time PCR is currently one of the promising methods to detect this infectious pathogen in clinical samples (6, 21, 26, 27). For successful development, optimization, and utilization of a PCR assay for detection of invasive candidiasis, an understanding of the kinetics of DNA released from C. albicans, alone and in the presence of host factors, is critical. We therefore studied the kinetics of DNA released from C. albicans by quantitative real-time PCR (qPCR) alone in vitro, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis.
MATERIALS AND METHODS
Primer and probe design. Utilizing fluorescence resonance energy transfer technology, a quantitative real-time PCR assay targeting the internal transcribed spacer (ITS) 1, 5.8S, and ITS2 regions of the rRNA gene complex was designed (Fig. 1). The primers and probes were based on a consensus sequence generated from multiple rRNA alignments of Candida species' sequences from GenBank, utilizing the Sequencher software package (Gene Codes Corp., Ann Arbor, MI) (Table 1). Initial primer and probe sequences were designed using the LightCycler probe design software version 2.0 (Idaho Technology, Inc., Salt Lake City, UT). The NCBI BLAST database search program was used to determine the primers' abilities to target the rRNA of C. albicans and, specifically, to not cross-hybridize with any mammalian DNA sequence. Once this was determined, primers and probes were further refined using Oligo Primer Analysis software, version 6.72 (Molecular Biology Insights, Cascade, CO) to minimize upper, lower, and upper/lower primer duplexes and hairpins and to optimize respective interactive melting temperatures of the primers and probes. The refined primers' sequence specificities were reconfirmed using the NCBI BLAST database search program. The amplicon generated was 218 bp in size.
PCR conditions. All PCR master mix setups were performed in a sterile biosafety cabinet located in a separate room from that where DNA extractions were performed. DNA extracted from samples was loaded into amplification vessels in a separate room from the PCR master mix setup. The PCR master mix consisted of 0.5 μM of each of the primers, 3 mM MgCl2, 0.025% bovine serum albumin (BSA; Sigma-Aldrich Corp., St. Louis, MO), 0.025 U/ml Platinum Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA), PCR 10x buffer (Invitrogen Corp., Carlsbad, CA), 0.2 mM PCR Nucleotide MixPLUS (1 dATP, dCTP, dGTP, and 3 dUTP; Roche Molecular Biochemicals, Indianapolis, IN), and 0.1 μM each of the fluorescein isothiocyanate (FITC) and LC Red-640 probes (RD640). In addition, HK-UNG thermostable uracil-N-glycosylase (Epicenter, Madison, WI) was utilized as recommended by the manufacturer to prevent potential amplicon carryover. Although the amplification system is considered a closed system, the reaction vessels are made of glass that has potential for breakage. The use of uracil-N-glycosylase ensures no amplicon contamination in such an event. The additional step also ensures that the quantitative assays are measuring only the desired template and not any potential amplicon. Each reaction contained a 5-μl aliquot of extracted specimen, together with 15 μl of the master mix. The cycling conditions were as follows. For one cycle: uracil activation, 37°C, 900 s; uracil heat inactivation, 95°C, 180 s. For 50 amplification cycles: denaturation, 95°C, 0 s (slope 20°C/s); annealing, 53°C, 5 s (slope 10°C/s); extension, 72°C, 15 s (slope 3°C/s). Quantitation standards, composed of 10-fold serial dilutions of C. albicans genomic DNA, were run in conjunction with each set of samples. For each PCR run a "kit blank" (water processed though extraction protocol) and a negative master mix control (water) were included. All amplification reactions were performed using the LightCycler 2.0 system (Roche Diagnostic Corp., Indianapolis, IN).
Analytical sensitivity and quantitation. Quantitation, accuracy, and precision of the real-time PCR assay were determined through serial dilutions of C. albicans genomic DNA. Candida albicans 8621 (ATCC MYA-1237) from a granulocytopenic patient with autopsy-proven disseminated candidiasis was used for all experiments. Organisms and culture were prepared as previously described (19). C. albicans DNA was extracted as described earlier (23). Briefly, an overnight culture of C. albicans was grown on Emmons' modified Sabouraud glucose agar plate (SGA) containing chloramphenicol and gentamicin (Bioworks, Inc., Baltimore, MD). Three isolated colonies of the organisms were subsequently suspended in 50 ml of Sabouraud dextrose medium (Emmons modification) (SDMEM; KD Medical, Columbia, MD) in 250-ml Erlenmeyer flasks and grown overnight at 37°C in a water bath to obtain blastoconidia in log-phase growth. One milliliter of overnight culture (5 x 107 to 1 x 108 cells) was harvested and washed with normal saline two times. Spheroplasts were prepared by resuspending the blastoconidia in 600 μl of spheroplast buffer containing 0.25 mg of lyticase (L-8137; Sigma, St. Louis, MO) per ml and incubated at 30°C for 45 min. Extraction of DNA from spheroplasts was performed using the QIAGEN DNeasy Plant Mini Kit (QIAGEN, Santa Clarita, CA). The standard protocol was followed, with the following two modifications: in step 2, 0.2 g of glass beads (G-8772; Sigma, St. Louis, MO) was added to each sample and vortexed at high speed for 10 min; in step 4, samples were centrifuged for 5 min at high speed (10,000 x g) before applying lysate to the QIAshredder spin column. DNA was eluted with 100 μl of 65°C preheated AE elution buffer. DNA was quantified using the Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Tenfold serial dilutions were made in triplicate, ranging from 2 x 105 fg to 2 x 10–1 fg, to test for sensitivity. Five microliters was loaded into each amplification reaction, resulting in a test range from 1 x 106 fg to 1 x 100 fg. These serial dilutions were used to assess efficiency of the assay, for sensitivity of the assay as well as for a standard curve for quantification (Fig. 2). Confirmation that the amplicon generated was from the intended target was performed utilizing the LightCycler data analysis software, version 3.5.28 (Roche Diagnostic Corp., Indianapolis, IN), by melting-curve profile analysis of all amplicons.
Specificity. Cross-reactivity of the assay was assessed by using DNA extracted from the following organisms: Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, Cryptococcus neoformans, Saccharomyces cerevisiae, Trichosporon asahii, Trichosporon inkin, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Pseudallescheria boydii, Penicillium marneffei, Penicillium chrysogenum, Penicillium notatum, Penicillium citrinum, Penicillium purpurogenum, rabbit (whole blood), and human (whole blood).
Inhibition studies. In order to confirm lack of PCR inhibitors from experimental samples, a separate set of PCR/fluorescence resonance energy transfer technology reactions was performed. A previously designed A. fumigatus-specific qPCR assay (17) was utilized to check for any signs of inhibition. Briefly, a master mix using the A. fumigatus primers/probes was made. The master mix was spiked with A. fumigatus genomic DNA. Five microliters of each experimental sample was added to 15 μl of this master mix. Additionally, 5 μl of water was added to 15 μl of the master mix as a reference for zero inhibition. Inhibition studies of all samples being analyzed were done in parallel to test for the presence or absence of inhibitors derived from either the samples themselves, the collection method of the samples, or carryover from the processing of the samples. The crossover values of the experimental samples were compared to the crossover values of the water samples. Presence of inhibition would result in a higher crossover value than those of water samples. Inhibition was not seen in any of the samples.
In vitro kinetics of extracellular DNA released from C. albicans. The inoculum was prepared as described above. After final washing, the blastoconidia were counted on a hemacytometer (Neubauer improved bright line Superior; Germany) and resuspended in the desired media to obtain the final concentration of 1 x 107 CFU/ml. Five milliters of the suspension was transferred to six-well flat-bottomed polypropylene plates (Costar 3527; Corning, New York) and incubated at 37°C and 5% CO2 on a rotary plate shaker (Heidolph Instruments, Germany). Media used included Hanks balanced salt solution (HBSS; Quality Biological, Inc., Gaithersburg, MD), HBSS plus 10% BSA (Sigma, St. Louis, MO), RPMI with L-glutamine (KD Medical, Columbia, MD), and RPMI plus 10% BSA. Samples were incubated for 0, 2, 4, 6, 8, 12, 16, and 24 h. At each time point, respective cultures were transferred to 15-ml polypropylene conical tubes (Falcon 2097; Becton Dickinson, Franklin Lakes, NJ), cells were pelleted by centrifuging at 800 x g, and supernatants were collected and stored at –30°C for later use.
In vitro kinetics of extracellular DNA released from C. albicans in the presence of human peripheral blood monocytes. Human peripheral blood monocytes from healthy donors were collected as described previously (13). Briefly, H-MNCs were isolated from healthy human donors by automated leukophoresis and counter-flow elutriation (J-6 M centrifuge; Beckman Instruments, Fullerton, CA). The isolated cell population was 95% monocytes, as determined by morphology and nonspecific esterase staining. The monocytes were washed once, resuspended in HBSS, and kept on ice until use. Monocytes (107 cells/ml HBSS) were incubated at an effector-to-target (E:T) ratio of 1:1 with 107 serum-opsonized C. albicans (107 cells/ml HBSS) in six-well flat-bottomed polypropylene plates. Control samples of C. albicans alone (107 cells/ml HBSS) were set up in parallel. An additional 1 ml HBSS was added to the control samples so total concentration would be equivalent to experimental samples. Samples were incubated for 0, 2, 4, 6, 8, 12, 16, and 24 h on a rotary plate shaker at 37°C and 5% CO2. Supernatants were collected as described above.
DNA extraction of fungal cell-free DNA. To avoid potential contamination, all DNA extractions were performed in a dead-air box (AirClean 600 PCR Workstation; AirClean Systems, Raleigh, NC). Extraction of fungal cell-free DNA was performed as described previously (27). In brief, 200 μl of the sample was processed using a QIAamp DNA Blood Mini Kit (QIAGEN, Santa Clarita, CA), with the following additions: each initial wash with buffer AW1 and buffer AW2 was performed twice. After application of the initial wash of each buffer, the buffer was allowed to set on the membrane for 5 min before spinning. Final elution was done with 200 μl of preheated (70°C) AE buffer. DNA extractions were stored at –30°C until samples were ready for quantification by qPCR.
DNA extraction of fungal cell-associated DNA. Extraction of fungal cell-associated DNA was performed using a DNeasy Tissue Kit (QIAGEN, Santa Clarita, CA) with the following modifications. Samples were pretreated with 100 μl spheroplast buffer (1.0 M sorbitol, 50.0 mM sodium phosphate monobasic, 0.1% 2-mercaptoethanol, 10 mg/ml lyticase [Sigma L-2524]) and 10 μl of lysing enzymes (Novozyme [Sigma L-1412], 20 mg/ml). Samples were vortexed vigorously and incubated at 37°C on a rotary plate shaker for 1 h. Two hundred eighty microliters of QiAmp Tissue Lysis Buffer and 20 μl of proteinase K were added and incubated at 55°C for 1 h in a thermomixer at 1,200 rpm. Next, 400 μl of QiAmp Lysis Buffer was added and incubated at 70°C for 10 min. Subsequently, 400 μl of ethyl alcohol (100%) was added and mixed thoroughly. Six hundred microliters was pipetted onto the minispin column and centrifuged at 6,000 x g. This was repeated with the remaining volume. Washes with AW1 and AW2 buffers were performed twice, with the initial wash of each allowed to set on the membrane for 5 min before centrifuging. A final elution was performed with 200 μl of preheated (70°C) AE buffer. DNA extractions were stored at –30°C until samples were ready for quantification by qPCR.
Fungal cell-associated DNA versus fungal cell-free DNA in blood fractions. An overnight culture of C. albicans in SDMEM was prepared as described above. One milliliter of whole blood from a normal rabbit was placed in a collection tube with K2-EDTA (Becton Dickinson, Franklin Lakes, NJ) and inoculated with 1 x 106 C. albicans blastoconidia and 1 x 106 fg of Aspergillus fumigatus genomic DNA. The application of A. fumigatus genomic DNA was used to clearly distinguish between fungal cell-associated DNA (for this experiment, C. albicans DNA) and fungal cell-free DNA (represented by A. fumigatus genomic DNA). The sample was mixed thoroughly and the volume was centrifuged at 500 x g at 4°C for 10 min. The plasma was then separated from the blood pellet. Both fungal cell-associated DNA and fungal cell-free DNA were extracted from 200 μl of each blood fraction (plasma and blood pellet) as described above. Levels of C. albicans DNA reflected fungal cell-associated DNA, while levels of A. fumigatus DNA reflected fungal cell-free DNA.
Animal inoculum. For preparation of the inoculum, three to five well-isolated colonies of C. albicans were sampled from a freshly grown culture SGA plate and suspended into 50 ml of SDMEM in a 250-ml Erlenmeyer flask. The suspension was incubated in a gyratory water bath at 80 oscillations per min at 37°C for 18 h. The suspension of C. albicans was then centrifuged at 3,000 x g for 10 min and washed three times with sterile normal saline (Quality Biological, Inc., Gaithersburg, MD). The concentration was adjusted by use of a hemacytometer and was confirmed by quantitative cultures of a 10-fold serial dilution. An inoculum of 1 x 106 blastoconidia suspended in a 5-ml volume of normal saline was slowly administered to each rabbit via the indwelling silastic central venous catheter on day 1 of the experiment. The inoculum size was confirmed by plating serial dilutions onto SGA plates. The pattern of infection of disseminated candidiasis permitted survival of nearly all rabbits throughout the experiment.
Animals. Female New Zealand White rabbits (Hazleton Research Products, Inc., Denver, PA) weighing 2.6 to 3.7 kg at the time of inoculation were used in experiments (n = 12). These studies were approved by the Animal Care and Use Committee of the National Cancer Institute. Rabbits were individually housed, maintained with water and standard rabbit feed ad libitum, and monitored under humane care and use in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, according to National Institutes of Health (NIH) guidelines for animal care, and in fulfillment of the guidelines of the National Research Council (16). Vascular access was established in each rabbit by the surgical placement of a silastic tunneled central venous catheter as previously described (28). The silastic catheter permitted nontraumatic venous access for repeated blood sampling for studies of biochemical and hematological parameters and administration of parenteral agents. Samples were drawn from all rabbits during the course of disseminated candidiasis. Rabbits were euthanized according to Animal Care and Use Committee-approved prespecified humane endpoints by intravenous administration of 65 mg of pentobarbital sodium/kg of body weight (pentobarbital sodium was in the form of 0.5 ml of Beuthanasia-D special [euthanasia solution]; Schering-Plough Animal Health Corp., Union, NJ) at the end of each experiment.
Animal blood cultures. One milliliter of whole blood was drawn into a 3-ml syringe (Becton Dickinson & Co., Franklin Lakes, NJ) and plated on an SGA plate. One milliliter of whole blood was drawn into a second 3-ml syringe and dispensed into 6-ml blood collection tubes with K2-EDTA. Samples were promptly centrifuged at 500 x g at 4°C for 10 min. Plasma was separated from the blood pellet and each fraction placed in separate microtubes (Sarstedt Inc., Newton, NC). To assess the presence or absence of viable organisms in each blood component, 0.1-ml aliquots of (each) plasma and blood were plated onto separate SGA plates. Culture plates were incubated at 37°C for 24 h, after which CFU were counted and the number of CFU/ml of blood was calculated for plasma and blood pellet. The method was sufficiently sensitive to detect 10 CFU/ml. The culture-negative plates were counted as 0 CFU/ml. The remaining plasma and blood pellet samples were stored at –30°C until further use.
DNA stability studies. The stability of cell-free C. albicans DNA in water, rabbit plasma, and human plasma was investigated at three temperatures (20°C, 4°C, and –30°C) over a period of 72 h. Cell-free C. albicans DNA (1 x 107 fg) was suspended in 200 μl of either water, rabbit plasma, or human plasma. A set was stored at each of the three temperatures stated. Extractions of cell-free DNA were performed at T = 0, 24, 48, and 72 h as previously mentioned. DNA levels were quantified using the C. albicans-specific qPCR assay described above.
Modeling and statistical and regression analyses. In vitro DNA kinetics data of C. albicans in HBSS ± 10% BSA and C. albicans in RPMI ± 10% BSA were analyzed using a Kruskal-Wallis test (nonparametric analysis of variance) from time zero h to 24 h. Values were expressed as means and standard errors of the means (SEM), and a P value of <0.01 was considered to be significant. In order to compare the entire DNA release curves in the presence and absence of H-MNCs, kinetics data were analyzed with a nonlinear regression analysis using the one-phase exponential association described by the equation E = Span x [1 – e^(–K x X)] + E0 where E is the concentration of DNA [log10(fg DNA/ml)] (dependent variable), X is the time in h (independent variable), E0 is E at time zero, Span is the increase of E, and K is a constant which can be used to calculate the DNA release rate equal to 0.633/K. Residuals were weighted by 1/SD2 where "SD" is the standard deviation. Goodness of fit was assessed with R2, SEM of fitted parameters and statistically significant deviation from the model was tested with the runs test. Best-fitted parameters of C. albicans DNA release curves in the presence and absence of H-MNCs were compared by t test (two-tailed P value). A P value of <0.001 was considered to be significant.
RESULTS
Analytical sensitivity and specificity. The sensitivity of the qPCR assay was validated through 10-fold serial dilutions of genomic DNA extracted from C. albicans. The assay consistently detected as low as 10 fg of C. albicans genomic DNA. The fluorescent signal was proportional to the log concentration of C. albicans genomic DNA (Fig. 2). The calculated PCR efficiency was 1.95. Specificity of the assay was tested against genomic DNA from a panel of organisms. The assay did not cross-react with any of the clinically relevant organisms or potential environmental contaminants tested.
In vitro kinetics of extracellular DNA released from C. albicans. Blastoconidia of C. albicans incubated in HBSS medium showed a significant release of DNA into the surrounding environment over a 24-h period (P = 0.001) (Fig. 3). Additional experiments incubating C. albicans blastoconidia in RPMI also showed a significant release of DNA into the surrounding environment over a 24-h period (P = 0.001) (Fig. 4). RPMI showed slightly increased levels of fungal cell-free DNA in the supernatant compared to HBSS. Addition of 10% BSA to either medium showed a trend toward increased levels of fungal cell-free DNA recovered from the supernatant.
In vitro DNA kinetics of extracellular DNA released from C. albicans in the presence of human peripheral blood monocytes. Serum-opsonized C. albicans blastoconidia, in the presence of H-MNCs, were observed to release a significantly greater amount of DNA into the extracellular fluid than that of C. albicans blastoconidia alone (P < 0.0001) (Fig. 5). Greater levels of fungal cell-free DNA in the media were observed after as little as 2 hours of incubation with H-MNCs. One-way analysis of variance showed a statistically significant difference in DNA release in the presence and the absence of H-MNCs after incubation periods of 8 h or longer. The one-phase exponential association model fit very well to the data with R2 > 0.98 and SEM < 0.5 log2, and no statistically significant deviation was found with the runs test. The best-fit parameters Span and K of DNA release curves were 2.5 and 0.3, respectively, in the absence of H-MNCs and 0.5 and 0.3, respectively, in the presence of H-MNCs. The difference in Span but not in K was statistically significant (P < 0.03 and P < 0.05, respectively).
Fungal cell-associated DNA versus fungal cell-free DNA in blood fractions. Fungal cell-free DNA was found at higher levels in plasma than in the blood pellet (P = 0.002) (Table 2). By comparison, fungal cell-associated DNA was found at greater levels in the blood pellet than in plasma (P = 0.002).
Quantitative blood cultures and DNA from a rabbit model of disseminated candidiasis. Blood cultures were performed on blood pellets from day 1 through day 8 (postinoculation) of the experiment. The yield of quantitative blood pellet cultures increased over the time course of the experiment (P = 0.0084, Mann-Whitney U test), reflecting the progression of disease during the experimental time course (Fig. 6). Cultures were also performed on plasma from day 1 through day 8 of the experiment. Plasma cultures were sporadic throughout the 8-day time course. DNA was extracted from the same plasma samples collected from day 1 through day 8 of the experiment. Mean DNA levels increased over the time course of the experiment (P = 0.0001, Mann-Whitney U test). The mean DNA level decreased on day 8 of the experiment, remaining statistically significant with respect to day 1 (P = 0.015, Mann-Whitney U test), demonstrating the release of genomic DNA of C. albicans into plasma during disseminated candidiasis. DNA was extracted from plasma obtained from normal rabbits (n = 6). All samples came up negative (data not shown).
DNA stability studies. Stability of cell-free C. albicans DNA in water, rabbit plasma, and human plasma was investigated over a 72-h period at three different temperatures (room temperature, 4°C, and –30°C). The cell-free DNA remained stable over 72 h regardless of storage temperatures tested or whether the DNA was suspended in water, rabbit plasma, or human plasma (Fig. 7A, B, and C, respectively).
DISCUSSION
This study investigated the kinetics of DNA released from C. albicans in vitro and in vivo using a qPCR assay specific for C. albicans. Genomic DNA was released from C. albicans into the extracellular fluid when incubated in standard media with or without additional protein. The presence of protein tended to increase the amount of genomic DNA from C. albicans found in the extracellular fluid. When incubated with human peripheral blood monocytes, there was a significantly greater release of DNA over time than that of C. albicans incubated alone, presumably in relationship to the damage inflicted by the phagocytic cells on fungal cells with subsequent release of DNA into the extracellular fluid. Consistent with the in vitro findings, fungal cell-free DNA was found at significantly high concentrations in plasma. These findings support the concept that genomic DNA of C. albicans is released into the extracellular fluid and that genomic DNA of C. albicans is found in high concentrations in plasma, despite the minimal presence of viable organisms present within the plasma component in nonneutropenic rabbits with experimental disseminated candidiasis. Collectively, these data indicate that plasma is an important source for circulating DNA of C. albicans and that these observations are compatible with the in vitro and in vivo kinetics of genomic DNA release from C. albicans over time.
To optimize molecular-based methods as potential diagnostic tools for detection of candidemia, a clearer understanding of the kinetics of genomic DNA of Candida is necessary. To this end, we initially developed a sensitive, species-specific, qPCR assay for DNA of C. albicans. The sensitivity of this assay was found to be 10 fg, which translated to a DNA content of less than one haploid genotype of C. albicans (7, 22). As with previous studies (2, 17, 29), use of the rRNA gene complex and, more specifically, the ITS region allowed for the design of a species-specific assay. Additionally, targeting a multicopy gene such as the rRNA gene increased the number of potential targets of the assay (12). Equally as important as sequence selection of the primers and probes for specificity were the cycling parameters used for amplification. Optimization of these parameters was performed to enhance specificity without compromising sensitivity or amplification efficiency.
By collecting the supernatants from media inoculated with C. albicans at different time points up to 24 h, extracting DNA, and performing qPCR, we were able to demonstrate that DNA is gradually released over time into the surrounding extracellular environment. The amount of fungal cell-free DNA measured in supernatants increased the longer the C. albicans cells were allowed to proliferate. The fungal cell-free DNA most likely reflected the natural life cycle of cell growth and death, upon which the DNA was released. These data demonstrate that genomic DNA of C. albicans is released into the extracellular fluid over the course of time in sparsely nutrient media or media enriched with protein or amino acids at relatively similar degrees. Despite vigorous centrifugation of Candida cells in removal of supernatant, an abundant amount of genomic DNA of C. albicans was present in a cell-free state.
Monocytes are known to be one of the initial phagocytic cell populations mediating host defenses against C. albicans (13). We hypothesized that phagocytic-mediated damage to Candida cells could result in increased release of genomic DNA. We therefore investigated the effect on the fungal cell-free DNA kinetics of exposing C. albicans to H-MNCs. Exposure of opsonized C. albicans blastoconidia to H-MNCs significantly increased the amount of genomic DNA released over 24 h compared to DNA released from opsonized C. albicans alone. This increase of genomic DNA release of H-MNC-exposed C. albicans over C. albicans alone was consistent with a direct effect of monocyte-mediated damage. Of note, we observed that the in vitro experiments with C. albicans alone and C. albicans plus H-MNCs showed a plateau of C. albicans DNA released into the extracellular environment. We believe this was a reflection of the in vitro experiments being a capacity-limited system. Classic cell growth curves plateau as nutrients in the media are depleted. The plateau of C. albicans DNA in the extracellular environment most likely reflects the plateau of C. albicans growth.
Following these experiments, we then studied which fraction of whole blood would be the best site for detecting fungal cell-free DNA. Currently there is no consensus on which blood fraction is best suited for detection of invasive candidiasis (14). Utilization of two species-specific assays (a C. albicans-specific qPCR assay and an A. fumigatus-specific qPCR assay) allowed us to differentiate between cell-associated DNA and cell-free DNA in our in vitro experiments. Whole blood was spiked with C. albicans cells (source of cell-associated DNA) and A. fumigatus genomic DNA (source of cell-free DNA). When assaying the extractions from the two blood components, any amplification seen using the C. albicans-specific assay would reflect cell-associated DNA from spiked C. albicans cells. Conversely, any amplification using the A. fumigatus-specific assay would reflect only cell-free DNA from the spiked genomic DNA of A. fumigatus. A greater proportion of fungal cell-associated DNA was found in the blood pellet fraction than in the plasma. The centrifugation used to separate the whole blood into its two fractions resulted in the denser, intact blastoconidia of C. albicans pelleting to the bottom of the tube with other cellular components of the whole blood. Conversely, fungal cell-free DNA was found in greater quantity in plasma than in the blood pellet. Based upon our in vitro kinetic data and the results demonstrating a partitioning of cell-free DNA into plasma, we hypothesized that plasma would be a useful source for detection of DNA from Candida albicans in a nonneutropenic rabbit model of disseminated candidiasis.
Samples of whole blood obtained from nonneutropenic rabbits with experimental disseminated candidiasis were collected daily and processed throughout the time course of the experiment. Due to the inhibitory effect of heparin on PCR (30), it was important to design our experiments such that the collection of all blood samples was performed in tubes with K2-EDTA as the anticoagulant. Increasing counts of CFU/ml of daily blood pellet cultures were clear indicators that infection had been well established and reflected the progression of disease from day 1 through day 8 of the experiment. Fungal cell-free DNA was found in the plasma fraction as early as day 1 postinoculation, with circulating fungal DNA levels increasing as progression of disease occurred. Unlike the in vitro models, the in vivo experiments were not capacity-limited systems. Therefore, blood cultures showed increased counts of CFU/ml over the time course of the experiment. Parallel levels of cell-free DNA in the plasma reflected the increased concentration of organisms found in the blood pellet. Additionally, the in vitro experiments showed the effect of H-MNCs on the DNA release of C. albicans. In vivo, there are many more host defense factors (e.g., neutrophils) that may play a role in releasing additional C. albicans DNA. Corresponding plasma cultures for circulating C. albicans remained very low to nonexistent, confirming that the fungal DNA detected in plasma was cell-free DNA. These findings are in agreement with previous studies which showed that dead or dying cells have been considered the source of extracellular DNA (4).
Once the DNA had been released from the cell, the issue of DNA stability in the extracellular environment needed to be ascertained. Our studies showed that C. albicans cell-free DNA remained stable in water and rabbit and human plasma over 72 h at various temperatures (room temperature, 4°C, and –30°C). These findings are in agreement with previous studies showing the stability of spiked A. fumigatus DNA in blood samples up to 72 h (11).
Although there have been studies that apply PCR assays to detection of deeply invasive candidiasis (2, 5, 12, 24-27), this to our knowledge is the first report that applies qPCR to investigate the kinetics of DNA release from C. albicans into the extracellular environment and, in addition, describes the role that H-MNCs play in Candida albicans DNA kinetics by increasing the release of fungal cell-free DNA. Further understanding of DNA kinetics of pathogens and the mechanisms by which DNA is released, especially those causing bloodstream infections, should improve strategies in designing and implementing molecular-based diagnostics for fungal pathogens.
ACKNOWLEDGMENTS
We thank Heidi A. Murray and Christine Mya-San for their assistance in conducting laboratory animal studies.
This research was supported by the Intramural Research Program of the NIH and National Cancer Institute.
REFERENCES
Benjamin, D. K., Jr., C. Poole, W. J. Steinbach, J. L. Rowen, and T. J. Walsh. 2003. Neonatal candidemia and end-organ damage: a critical appraisal of the literature using meta-analytic techniques. Pediatrics 112:634-640.
Bu, R., R. K. Sathiapalan, M. M. Ibrahim, I. Al-Mohsen, E. Almodavar, M. I. Gutierrez, and K. Bhatia. 2005. Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus. J. Med. Microbiol. 54:243-248.
Buchman, T. G., M. Rossier, W. G. Merz, and P. Charache. 1990. Detection of surgical pathogens by in vitro DNA amplification. Part I: rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene. Surgery 108:338-346.
Choi, J. J., C. F. Reich III, and D. S. Pisetsky. 2004. Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro. Scand. J. Immunol. 60:159-166.
Chryssanthou, E., B. Andersson, B. Petrini, S. Lofdahl, and J. Tollemar. 1994. Detection of Candida albicans DNA in serum by polymerase chain reaction. Scand. J. Infect. Dis. 26:479-485.
Ellepola, A. N., and C. J. Morrison. 2005. Laboratory diagnosis of invasive candidiasis. J. Microbiol. 43:65-84.
Fleischmann, J., and D. H. Howard. 1988. The DNA content of nongerminative variants of Candida albicans. Nucleic Acids Res. 16:765.
Ghannoum, M. A. 2001. Candida: a causative agent of an emerging infection. J. Investig. Dermatol. Symp. Proc. 6:188-196.
Gudlaugsson, O., S. Gillespie, K. Lee, J. Vande Berg, J. Hu, S. Messer, L. Herwaldt, M. Pfaller, and D. Diekema. 2003. Attributable mortality of nosocomial candidemia, revisited. Clin. Infect. Dis. 37:1172-1177.
Hajjeh, R. A., A. N. Sofair, L. H. Harrison, G. M. Lyon, B. A. Arthington-Skaggs, S. A. Mirza, M. Phelan, J. Morgan, W. Lee-Yang, M. A. Ciblak, L. E. Benjamin, L. T. Sanza, S. Huie, S. F. Yeo, M. E. Brandt, and D. W. Warnock. 2004. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J. Clin. Microbiol. 42:1519-1527.
Hebart, H., J. Loffler, C. Meisner, F. Serey, D. Schmidt, A. Bohme, H. Martin, A. Engel, D. Bunje, W. V. Kern, U. Schumacher, L. Kanz, and H. Einsele. 2000. Early detection of aspergillus infection after allogeneic stem cell transplantation by polymerase chain reaction screening. J. Infect. Dis. 181:1713-1719.
Hopfer, R. L., P. Walden, S. Setterquist, and W. E. Highsmith. 1993. Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis. J. Med. Vet. Mycol. 31:65-75.
Kim, H. S., E. H. Choi, J. Khan, E. Roilides, A. Francesconi, M. Kasai, T. Sein, R. L. Schaufele, K. Sakurai, C. G. Son, B. T. Greer, S. Chanock, C. A. Lyman, and T. J. Walsh. 2005. Expression of genes encoding innate host defense molecules in normal human monocytes in response to Candida albicans. Infect. Immun. 73:3714-3724.
Maaroufi, Y., N. Ahariz, M. Husson, and F. Crokaert. 2004. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay. J. Clin. Microbiol. 42:3159-3163.
Miller, L. G., R. A. Hajjeh, and J. E. Edwards, Jr. 2001. Estimating the cost of nosocomial candidemia in the United States. Clin. Infect. Dis. 32:1110.
National Research Council. 1996. Guide for the care and use of laboratory animals. National Academy Press, Washington, D.C.
O'Sullivan, C. E., M. Kasai, A. Francesconi, V. Petraitis, R. Petraitiene, A. M. Kelaher, A. A. Sarafandi, and T. J. Walsh. 2003. Development and validation of a quantitative real-time PCR assay using fluorescence resonance energy transfer technology for detection of Aspergillus fumigatus in experimental invasive pulmonary aspergillosis. J. Clin. Microbiol. 41:5676-5682.
Pappas, P. G., J. H. Rex, J. Lee, R. J. Hamill, R. A. Larsen, W. Powderly, C. A. Kauffman, N. Hyslop, J. E. Mangino, S. Chapman, H. W. Horowitz, J. E. Edwards, and W. E. Dismukes. 2003. A prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients. Clin. Infect. Dis. 37:634-643.
Petraitis, V., R. Petraitiene, A. H. Groll, K. Roussillon, M. Hemmings, C. A. Lyman, T. Sein, J. Bacher, I. Bekersky, and T. J. Walsh. 2002. Comparative antifungal activities and plasma pharmacokinetics of micafungin (FK463) against disseminated candidiasis and invasive pulmonary aspergillosis in persistently neutropenic rabbits. Antimicrob. Agents Chemother. 46:1857-1869.
Pfaller, M. A., and D. J. Diekema. 2004. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42:4419-4431.
Reiss, E., T. Obayashi, K. Orle, M. Yoshida, and R. M. Zancope-Oliveira. 2000. Non-culture based diagnostic tests for mycotic infections. Med. Mycol. 38(Suppl. 1):147-159.
Riggsby, W. S., L. J. Torres-Bauza, J. W. Wills, and T. M. Townes. 1982. DNA content, kinetic complexity, and the ploidy question in Candida albicans. Mol. Cell. Biol. 2:853-862.
Roilides, E., E. Farmaki, J. Evdoridou, A. Francesconi, M. Kasai, J. Filioti, M. Tsivitanidou, D. Sofianou, G. Kremenopoulos, and T. J. Walsh. 2003. Candida tropicalis in a neonatal intensive care unit: epidemiologic and molecular analysis of an outbreak of infection with an uncommon neonatal pathogen. J. Clin. Microbiol. 41:735-741.
Sakai, T., K. Ikegami, E. Yoshinaga, R. Uesugi-Hayakawa, and A. Wakizaka. 2000. Rapid, sensitive and simple detection of candida deep mycosis by amplification of 18S ribosomal RNA gene; comparison with assay of serum beta-D-glucan level in clinical samples. Tohoku J. Exp. Med. 190:119-128.
Skladny, H., D. Buchheidt, C. Baust, F. Krieg-Schneider, W. Seifarth, C. Leib-Msch, and R. Hehlmann. 1999. Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR. J. Clin. Microbiol. 37:3865-3871.
Trama, J. P., E. Mordechai, and M. E. Adelson. 2005. Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing. Mol. Cell. Probes 19:145-152.
Wahyuningsih, R., H.-J. Freisleben, H.-G. Sonntag, and P. Schnitzler. 2000. Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis. J. Clin. Microbiol. 38:3016-3021.
Walsh, T. J., J. Bacher, and P. A. Pizzo. 1988. Chronic silastic central venous catheterization for induction, maintenance and support of persistent granulocytopenia in rabbits. Lab. Anim. Sci. 38:467-471.
Williamson, E. C., and J. P. Leeming. 1999. Molecular approaches for the diagnosis and epidemiological investigation of Aspergillus infection. Mycoses 42(Suppl. 2):7-10.
Woo, P. C., S. K. Lo, K. Y. Yuen, J. S. Peiris, H. Siau, E. K. Chiu, R. H. Liang, and T. K. Chan. 1997. Detection of CMV DNA in bone marrow transplant recipients: plasma versus leucocyte polymerase chain reaction. J. Clin. Pathol. 50:231-235.(Miki Kasai, Andrea France)
SAIC-Frederick, Inc., Frederick, Maryland
Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, Maryland
ABSTRACT
Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of C. albicans incubated in Hanks balanced salt solution (±10% bovine serum albumin [BSA]) or RPMI (±10% BSA) showed a significant release of DNA at T equal to 24 h compared to T equal to 0 h (P 0.01). C. albicans incubated with H-MNCs exhibited a greater release of DNA than C. albicans cells alone over 24 h (P = 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of C. albicans, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by C. albicans collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of C. albicans DNA.
INTRODUCTION
Invasive candidiasis remains one of the leading causes of morbidity and mortality in immunocompromised and critically ill patients (1, 8, 9, 15, 18, 20, 23), with Candida albicans infections comprising 45% of all Candida bloodstream infections (10). Since the earliest introduction of PCR for the detection of C. albicans in blood (3, 12), this technology has been increasingly refined for molecular detection of deeply invasive candidiasis. Real-time PCR is currently one of the promising methods to detect this infectious pathogen in clinical samples (6, 21, 26, 27). For successful development, optimization, and utilization of a PCR assay for detection of invasive candidiasis, an understanding of the kinetics of DNA released from C. albicans, alone and in the presence of host factors, is critical. We therefore studied the kinetics of DNA released from C. albicans by quantitative real-time PCR (qPCR) alone in vitro, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis.
MATERIALS AND METHODS
Primer and probe design. Utilizing fluorescence resonance energy transfer technology, a quantitative real-time PCR assay targeting the internal transcribed spacer (ITS) 1, 5.8S, and ITS2 regions of the rRNA gene complex was designed (Fig. 1). The primers and probes were based on a consensus sequence generated from multiple rRNA alignments of Candida species' sequences from GenBank, utilizing the Sequencher software package (Gene Codes Corp., Ann Arbor, MI) (Table 1). Initial primer and probe sequences were designed using the LightCycler probe design software version 2.0 (Idaho Technology, Inc., Salt Lake City, UT). The NCBI BLAST database search program was used to determine the primers' abilities to target the rRNA of C. albicans and, specifically, to not cross-hybridize with any mammalian DNA sequence. Once this was determined, primers and probes were further refined using Oligo Primer Analysis software, version 6.72 (Molecular Biology Insights, Cascade, CO) to minimize upper, lower, and upper/lower primer duplexes and hairpins and to optimize respective interactive melting temperatures of the primers and probes. The refined primers' sequence specificities were reconfirmed using the NCBI BLAST database search program. The amplicon generated was 218 bp in size.
PCR conditions. All PCR master mix setups were performed in a sterile biosafety cabinet located in a separate room from that where DNA extractions were performed. DNA extracted from samples was loaded into amplification vessels in a separate room from the PCR master mix setup. The PCR master mix consisted of 0.5 μM of each of the primers, 3 mM MgCl2, 0.025% bovine serum albumin (BSA; Sigma-Aldrich Corp., St. Louis, MO), 0.025 U/ml Platinum Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA), PCR 10x buffer (Invitrogen Corp., Carlsbad, CA), 0.2 mM PCR Nucleotide MixPLUS (1 dATP, dCTP, dGTP, and 3 dUTP; Roche Molecular Biochemicals, Indianapolis, IN), and 0.1 μM each of the fluorescein isothiocyanate (FITC) and LC Red-640 probes (RD640). In addition, HK-UNG thermostable uracil-N-glycosylase (Epicenter, Madison, WI) was utilized as recommended by the manufacturer to prevent potential amplicon carryover. Although the amplification system is considered a closed system, the reaction vessels are made of glass that has potential for breakage. The use of uracil-N-glycosylase ensures no amplicon contamination in such an event. The additional step also ensures that the quantitative assays are measuring only the desired template and not any potential amplicon. Each reaction contained a 5-μl aliquot of extracted specimen, together with 15 μl of the master mix. The cycling conditions were as follows. For one cycle: uracil activation, 37°C, 900 s; uracil heat inactivation, 95°C, 180 s. For 50 amplification cycles: denaturation, 95°C, 0 s (slope 20°C/s); annealing, 53°C, 5 s (slope 10°C/s); extension, 72°C, 15 s (slope 3°C/s). Quantitation standards, composed of 10-fold serial dilutions of C. albicans genomic DNA, were run in conjunction with each set of samples. For each PCR run a "kit blank" (water processed though extraction protocol) and a negative master mix control (water) were included. All amplification reactions were performed using the LightCycler 2.0 system (Roche Diagnostic Corp., Indianapolis, IN).
Analytical sensitivity and quantitation. Quantitation, accuracy, and precision of the real-time PCR assay were determined through serial dilutions of C. albicans genomic DNA. Candida albicans 8621 (ATCC MYA-1237) from a granulocytopenic patient with autopsy-proven disseminated candidiasis was used for all experiments. Organisms and culture were prepared as previously described (19). C. albicans DNA was extracted as described earlier (23). Briefly, an overnight culture of C. albicans was grown on Emmons' modified Sabouraud glucose agar plate (SGA) containing chloramphenicol and gentamicin (Bioworks, Inc., Baltimore, MD). Three isolated colonies of the organisms were subsequently suspended in 50 ml of Sabouraud dextrose medium (Emmons modification) (SDMEM; KD Medical, Columbia, MD) in 250-ml Erlenmeyer flasks and grown overnight at 37°C in a water bath to obtain blastoconidia in log-phase growth. One milliliter of overnight culture (5 x 107 to 1 x 108 cells) was harvested and washed with normal saline two times. Spheroplasts were prepared by resuspending the blastoconidia in 600 μl of spheroplast buffer containing 0.25 mg of lyticase (L-8137; Sigma, St. Louis, MO) per ml and incubated at 30°C for 45 min. Extraction of DNA from spheroplasts was performed using the QIAGEN DNeasy Plant Mini Kit (QIAGEN, Santa Clarita, CA). The standard protocol was followed, with the following two modifications: in step 2, 0.2 g of glass beads (G-8772; Sigma, St. Louis, MO) was added to each sample and vortexed at high speed for 10 min; in step 4, samples were centrifuged for 5 min at high speed (10,000 x g) before applying lysate to the QIAshredder spin column. DNA was eluted with 100 μl of 65°C preheated AE elution buffer. DNA was quantified using the Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Tenfold serial dilutions were made in triplicate, ranging from 2 x 105 fg to 2 x 10–1 fg, to test for sensitivity. Five microliters was loaded into each amplification reaction, resulting in a test range from 1 x 106 fg to 1 x 100 fg. These serial dilutions were used to assess efficiency of the assay, for sensitivity of the assay as well as for a standard curve for quantification (Fig. 2). Confirmation that the amplicon generated was from the intended target was performed utilizing the LightCycler data analysis software, version 3.5.28 (Roche Diagnostic Corp., Indianapolis, IN), by melting-curve profile analysis of all amplicons.
Specificity. Cross-reactivity of the assay was assessed by using DNA extracted from the following organisms: Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, Cryptococcus neoformans, Saccharomyces cerevisiae, Trichosporon asahii, Trichosporon inkin, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Pseudallescheria boydii, Penicillium marneffei, Penicillium chrysogenum, Penicillium notatum, Penicillium citrinum, Penicillium purpurogenum, rabbit (whole blood), and human (whole blood).
Inhibition studies. In order to confirm lack of PCR inhibitors from experimental samples, a separate set of PCR/fluorescence resonance energy transfer technology reactions was performed. A previously designed A. fumigatus-specific qPCR assay (17) was utilized to check for any signs of inhibition. Briefly, a master mix using the A. fumigatus primers/probes was made. The master mix was spiked with A. fumigatus genomic DNA. Five microliters of each experimental sample was added to 15 μl of this master mix. Additionally, 5 μl of water was added to 15 μl of the master mix as a reference for zero inhibition. Inhibition studies of all samples being analyzed were done in parallel to test for the presence or absence of inhibitors derived from either the samples themselves, the collection method of the samples, or carryover from the processing of the samples. The crossover values of the experimental samples were compared to the crossover values of the water samples. Presence of inhibition would result in a higher crossover value than those of water samples. Inhibition was not seen in any of the samples.
In vitro kinetics of extracellular DNA released from C. albicans. The inoculum was prepared as described above. After final washing, the blastoconidia were counted on a hemacytometer (Neubauer improved bright line Superior; Germany) and resuspended in the desired media to obtain the final concentration of 1 x 107 CFU/ml. Five milliters of the suspension was transferred to six-well flat-bottomed polypropylene plates (Costar 3527; Corning, New York) and incubated at 37°C and 5% CO2 on a rotary plate shaker (Heidolph Instruments, Germany). Media used included Hanks balanced salt solution (HBSS; Quality Biological, Inc., Gaithersburg, MD), HBSS plus 10% BSA (Sigma, St. Louis, MO), RPMI with L-glutamine (KD Medical, Columbia, MD), and RPMI plus 10% BSA. Samples were incubated for 0, 2, 4, 6, 8, 12, 16, and 24 h. At each time point, respective cultures were transferred to 15-ml polypropylene conical tubes (Falcon 2097; Becton Dickinson, Franklin Lakes, NJ), cells were pelleted by centrifuging at 800 x g, and supernatants were collected and stored at –30°C for later use.
In vitro kinetics of extracellular DNA released from C. albicans in the presence of human peripheral blood monocytes. Human peripheral blood monocytes from healthy donors were collected as described previously (13). Briefly, H-MNCs were isolated from healthy human donors by automated leukophoresis and counter-flow elutriation (J-6 M centrifuge; Beckman Instruments, Fullerton, CA). The isolated cell population was 95% monocytes, as determined by morphology and nonspecific esterase staining. The monocytes were washed once, resuspended in HBSS, and kept on ice until use. Monocytes (107 cells/ml HBSS) were incubated at an effector-to-target (E:T) ratio of 1:1 with 107 serum-opsonized C. albicans (107 cells/ml HBSS) in six-well flat-bottomed polypropylene plates. Control samples of C. albicans alone (107 cells/ml HBSS) were set up in parallel. An additional 1 ml HBSS was added to the control samples so total concentration would be equivalent to experimental samples. Samples were incubated for 0, 2, 4, 6, 8, 12, 16, and 24 h on a rotary plate shaker at 37°C and 5% CO2. Supernatants were collected as described above.
DNA extraction of fungal cell-free DNA. To avoid potential contamination, all DNA extractions were performed in a dead-air box (AirClean 600 PCR Workstation; AirClean Systems, Raleigh, NC). Extraction of fungal cell-free DNA was performed as described previously (27). In brief, 200 μl of the sample was processed using a QIAamp DNA Blood Mini Kit (QIAGEN, Santa Clarita, CA), with the following additions: each initial wash with buffer AW1 and buffer AW2 was performed twice. After application of the initial wash of each buffer, the buffer was allowed to set on the membrane for 5 min before spinning. Final elution was done with 200 μl of preheated (70°C) AE buffer. DNA extractions were stored at –30°C until samples were ready for quantification by qPCR.
DNA extraction of fungal cell-associated DNA. Extraction of fungal cell-associated DNA was performed using a DNeasy Tissue Kit (QIAGEN, Santa Clarita, CA) with the following modifications. Samples were pretreated with 100 μl spheroplast buffer (1.0 M sorbitol, 50.0 mM sodium phosphate monobasic, 0.1% 2-mercaptoethanol, 10 mg/ml lyticase [Sigma L-2524]) and 10 μl of lysing enzymes (Novozyme [Sigma L-1412], 20 mg/ml). Samples were vortexed vigorously and incubated at 37°C on a rotary plate shaker for 1 h. Two hundred eighty microliters of QiAmp Tissue Lysis Buffer and 20 μl of proteinase K were added and incubated at 55°C for 1 h in a thermomixer at 1,200 rpm. Next, 400 μl of QiAmp Lysis Buffer was added and incubated at 70°C for 10 min. Subsequently, 400 μl of ethyl alcohol (100%) was added and mixed thoroughly. Six hundred microliters was pipetted onto the minispin column and centrifuged at 6,000 x g. This was repeated with the remaining volume. Washes with AW1 and AW2 buffers were performed twice, with the initial wash of each allowed to set on the membrane for 5 min before centrifuging. A final elution was performed with 200 μl of preheated (70°C) AE buffer. DNA extractions were stored at –30°C until samples were ready for quantification by qPCR.
Fungal cell-associated DNA versus fungal cell-free DNA in blood fractions. An overnight culture of C. albicans in SDMEM was prepared as described above. One milliliter of whole blood from a normal rabbit was placed in a collection tube with K2-EDTA (Becton Dickinson, Franklin Lakes, NJ) and inoculated with 1 x 106 C. albicans blastoconidia and 1 x 106 fg of Aspergillus fumigatus genomic DNA. The application of A. fumigatus genomic DNA was used to clearly distinguish between fungal cell-associated DNA (for this experiment, C. albicans DNA) and fungal cell-free DNA (represented by A. fumigatus genomic DNA). The sample was mixed thoroughly and the volume was centrifuged at 500 x g at 4°C for 10 min. The plasma was then separated from the blood pellet. Both fungal cell-associated DNA and fungal cell-free DNA were extracted from 200 μl of each blood fraction (plasma and blood pellet) as described above. Levels of C. albicans DNA reflected fungal cell-associated DNA, while levels of A. fumigatus DNA reflected fungal cell-free DNA.
Animal inoculum. For preparation of the inoculum, three to five well-isolated colonies of C. albicans were sampled from a freshly grown culture SGA plate and suspended into 50 ml of SDMEM in a 250-ml Erlenmeyer flask. The suspension was incubated in a gyratory water bath at 80 oscillations per min at 37°C for 18 h. The suspension of C. albicans was then centrifuged at 3,000 x g for 10 min and washed three times with sterile normal saline (Quality Biological, Inc., Gaithersburg, MD). The concentration was adjusted by use of a hemacytometer and was confirmed by quantitative cultures of a 10-fold serial dilution. An inoculum of 1 x 106 blastoconidia suspended in a 5-ml volume of normal saline was slowly administered to each rabbit via the indwelling silastic central venous catheter on day 1 of the experiment. The inoculum size was confirmed by plating serial dilutions onto SGA plates. The pattern of infection of disseminated candidiasis permitted survival of nearly all rabbits throughout the experiment.
Animals. Female New Zealand White rabbits (Hazleton Research Products, Inc., Denver, PA) weighing 2.6 to 3.7 kg at the time of inoculation were used in experiments (n = 12). These studies were approved by the Animal Care and Use Committee of the National Cancer Institute. Rabbits were individually housed, maintained with water and standard rabbit feed ad libitum, and monitored under humane care and use in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, according to National Institutes of Health (NIH) guidelines for animal care, and in fulfillment of the guidelines of the National Research Council (16). Vascular access was established in each rabbit by the surgical placement of a silastic tunneled central venous catheter as previously described (28). The silastic catheter permitted nontraumatic venous access for repeated blood sampling for studies of biochemical and hematological parameters and administration of parenteral agents. Samples were drawn from all rabbits during the course of disseminated candidiasis. Rabbits were euthanized according to Animal Care and Use Committee-approved prespecified humane endpoints by intravenous administration of 65 mg of pentobarbital sodium/kg of body weight (pentobarbital sodium was in the form of 0.5 ml of Beuthanasia-D special [euthanasia solution]; Schering-Plough Animal Health Corp., Union, NJ) at the end of each experiment.
Animal blood cultures. One milliliter of whole blood was drawn into a 3-ml syringe (Becton Dickinson & Co., Franklin Lakes, NJ) and plated on an SGA plate. One milliliter of whole blood was drawn into a second 3-ml syringe and dispensed into 6-ml blood collection tubes with K2-EDTA. Samples were promptly centrifuged at 500 x g at 4°C for 10 min. Plasma was separated from the blood pellet and each fraction placed in separate microtubes (Sarstedt Inc., Newton, NC). To assess the presence or absence of viable organisms in each blood component, 0.1-ml aliquots of (each) plasma and blood were plated onto separate SGA plates. Culture plates were incubated at 37°C for 24 h, after which CFU were counted and the number of CFU/ml of blood was calculated for plasma and blood pellet. The method was sufficiently sensitive to detect 10 CFU/ml. The culture-negative plates were counted as 0 CFU/ml. The remaining plasma and blood pellet samples were stored at –30°C until further use.
DNA stability studies. The stability of cell-free C. albicans DNA in water, rabbit plasma, and human plasma was investigated at three temperatures (20°C, 4°C, and –30°C) over a period of 72 h. Cell-free C. albicans DNA (1 x 107 fg) was suspended in 200 μl of either water, rabbit plasma, or human plasma. A set was stored at each of the three temperatures stated. Extractions of cell-free DNA were performed at T = 0, 24, 48, and 72 h as previously mentioned. DNA levels were quantified using the C. albicans-specific qPCR assay described above.
Modeling and statistical and regression analyses. In vitro DNA kinetics data of C. albicans in HBSS ± 10% BSA and C. albicans in RPMI ± 10% BSA were analyzed using a Kruskal-Wallis test (nonparametric analysis of variance) from time zero h to 24 h. Values were expressed as means and standard errors of the means (SEM), and a P value of <0.01 was considered to be significant. In order to compare the entire DNA release curves in the presence and absence of H-MNCs, kinetics data were analyzed with a nonlinear regression analysis using the one-phase exponential association described by the equation E = Span x [1 – e^(–K x X)] + E0 where E is the concentration of DNA [log10(fg DNA/ml)] (dependent variable), X is the time in h (independent variable), E0 is E at time zero, Span is the increase of E, and K is a constant which can be used to calculate the DNA release rate equal to 0.633/K. Residuals were weighted by 1/SD2 where "SD" is the standard deviation. Goodness of fit was assessed with R2, SEM of fitted parameters and statistically significant deviation from the model was tested with the runs test. Best-fitted parameters of C. albicans DNA release curves in the presence and absence of H-MNCs were compared by t test (two-tailed P value). A P value of <0.001 was considered to be significant.
RESULTS
Analytical sensitivity and specificity. The sensitivity of the qPCR assay was validated through 10-fold serial dilutions of genomic DNA extracted from C. albicans. The assay consistently detected as low as 10 fg of C. albicans genomic DNA. The fluorescent signal was proportional to the log concentration of C. albicans genomic DNA (Fig. 2). The calculated PCR efficiency was 1.95. Specificity of the assay was tested against genomic DNA from a panel of organisms. The assay did not cross-react with any of the clinically relevant organisms or potential environmental contaminants tested.
In vitro kinetics of extracellular DNA released from C. albicans. Blastoconidia of C. albicans incubated in HBSS medium showed a significant release of DNA into the surrounding environment over a 24-h period (P = 0.001) (Fig. 3). Additional experiments incubating C. albicans blastoconidia in RPMI also showed a significant release of DNA into the surrounding environment over a 24-h period (P = 0.001) (Fig. 4). RPMI showed slightly increased levels of fungal cell-free DNA in the supernatant compared to HBSS. Addition of 10% BSA to either medium showed a trend toward increased levels of fungal cell-free DNA recovered from the supernatant.
In vitro DNA kinetics of extracellular DNA released from C. albicans in the presence of human peripheral blood monocytes. Serum-opsonized C. albicans blastoconidia, in the presence of H-MNCs, were observed to release a significantly greater amount of DNA into the extracellular fluid than that of C. albicans blastoconidia alone (P < 0.0001) (Fig. 5). Greater levels of fungal cell-free DNA in the media were observed after as little as 2 hours of incubation with H-MNCs. One-way analysis of variance showed a statistically significant difference in DNA release in the presence and the absence of H-MNCs after incubation periods of 8 h or longer. The one-phase exponential association model fit very well to the data with R2 > 0.98 and SEM < 0.5 log2, and no statistically significant deviation was found with the runs test. The best-fit parameters Span and K of DNA release curves were 2.5 and 0.3, respectively, in the absence of H-MNCs and 0.5 and 0.3, respectively, in the presence of H-MNCs. The difference in Span but not in K was statistically significant (P < 0.03 and P < 0.05, respectively).
Fungal cell-associated DNA versus fungal cell-free DNA in blood fractions. Fungal cell-free DNA was found at higher levels in plasma than in the blood pellet (P = 0.002) (Table 2). By comparison, fungal cell-associated DNA was found at greater levels in the blood pellet than in plasma (P = 0.002).
Quantitative blood cultures and DNA from a rabbit model of disseminated candidiasis. Blood cultures were performed on blood pellets from day 1 through day 8 (postinoculation) of the experiment. The yield of quantitative blood pellet cultures increased over the time course of the experiment (P = 0.0084, Mann-Whitney U test), reflecting the progression of disease during the experimental time course (Fig. 6). Cultures were also performed on plasma from day 1 through day 8 of the experiment. Plasma cultures were sporadic throughout the 8-day time course. DNA was extracted from the same plasma samples collected from day 1 through day 8 of the experiment. Mean DNA levels increased over the time course of the experiment (P = 0.0001, Mann-Whitney U test). The mean DNA level decreased on day 8 of the experiment, remaining statistically significant with respect to day 1 (P = 0.015, Mann-Whitney U test), demonstrating the release of genomic DNA of C. albicans into plasma during disseminated candidiasis. DNA was extracted from plasma obtained from normal rabbits (n = 6). All samples came up negative (data not shown).
DNA stability studies. Stability of cell-free C. albicans DNA in water, rabbit plasma, and human plasma was investigated over a 72-h period at three different temperatures (room temperature, 4°C, and –30°C). The cell-free DNA remained stable over 72 h regardless of storage temperatures tested or whether the DNA was suspended in water, rabbit plasma, or human plasma (Fig. 7A, B, and C, respectively).
DISCUSSION
This study investigated the kinetics of DNA released from C. albicans in vitro and in vivo using a qPCR assay specific for C. albicans. Genomic DNA was released from C. albicans into the extracellular fluid when incubated in standard media with or without additional protein. The presence of protein tended to increase the amount of genomic DNA from C. albicans found in the extracellular fluid. When incubated with human peripheral blood monocytes, there was a significantly greater release of DNA over time than that of C. albicans incubated alone, presumably in relationship to the damage inflicted by the phagocytic cells on fungal cells with subsequent release of DNA into the extracellular fluid. Consistent with the in vitro findings, fungal cell-free DNA was found at significantly high concentrations in plasma. These findings support the concept that genomic DNA of C. albicans is released into the extracellular fluid and that genomic DNA of C. albicans is found in high concentrations in plasma, despite the minimal presence of viable organisms present within the plasma component in nonneutropenic rabbits with experimental disseminated candidiasis. Collectively, these data indicate that plasma is an important source for circulating DNA of C. albicans and that these observations are compatible with the in vitro and in vivo kinetics of genomic DNA release from C. albicans over time.
To optimize molecular-based methods as potential diagnostic tools for detection of candidemia, a clearer understanding of the kinetics of genomic DNA of Candida is necessary. To this end, we initially developed a sensitive, species-specific, qPCR assay for DNA of C. albicans. The sensitivity of this assay was found to be 10 fg, which translated to a DNA content of less than one haploid genotype of C. albicans (7, 22). As with previous studies (2, 17, 29), use of the rRNA gene complex and, more specifically, the ITS region allowed for the design of a species-specific assay. Additionally, targeting a multicopy gene such as the rRNA gene increased the number of potential targets of the assay (12). Equally as important as sequence selection of the primers and probes for specificity were the cycling parameters used for amplification. Optimization of these parameters was performed to enhance specificity without compromising sensitivity or amplification efficiency.
By collecting the supernatants from media inoculated with C. albicans at different time points up to 24 h, extracting DNA, and performing qPCR, we were able to demonstrate that DNA is gradually released over time into the surrounding extracellular environment. The amount of fungal cell-free DNA measured in supernatants increased the longer the C. albicans cells were allowed to proliferate. The fungal cell-free DNA most likely reflected the natural life cycle of cell growth and death, upon which the DNA was released. These data demonstrate that genomic DNA of C. albicans is released into the extracellular fluid over the course of time in sparsely nutrient media or media enriched with protein or amino acids at relatively similar degrees. Despite vigorous centrifugation of Candida cells in removal of supernatant, an abundant amount of genomic DNA of C. albicans was present in a cell-free state.
Monocytes are known to be one of the initial phagocytic cell populations mediating host defenses against C. albicans (13). We hypothesized that phagocytic-mediated damage to Candida cells could result in increased release of genomic DNA. We therefore investigated the effect on the fungal cell-free DNA kinetics of exposing C. albicans to H-MNCs. Exposure of opsonized C. albicans blastoconidia to H-MNCs significantly increased the amount of genomic DNA released over 24 h compared to DNA released from opsonized C. albicans alone. This increase of genomic DNA release of H-MNC-exposed C. albicans over C. albicans alone was consistent with a direct effect of monocyte-mediated damage. Of note, we observed that the in vitro experiments with C. albicans alone and C. albicans plus H-MNCs showed a plateau of C. albicans DNA released into the extracellular environment. We believe this was a reflection of the in vitro experiments being a capacity-limited system. Classic cell growth curves plateau as nutrients in the media are depleted. The plateau of C. albicans DNA in the extracellular environment most likely reflects the plateau of C. albicans growth.
Following these experiments, we then studied which fraction of whole blood would be the best site for detecting fungal cell-free DNA. Currently there is no consensus on which blood fraction is best suited for detection of invasive candidiasis (14). Utilization of two species-specific assays (a C. albicans-specific qPCR assay and an A. fumigatus-specific qPCR assay) allowed us to differentiate between cell-associated DNA and cell-free DNA in our in vitro experiments. Whole blood was spiked with C. albicans cells (source of cell-associated DNA) and A. fumigatus genomic DNA (source of cell-free DNA). When assaying the extractions from the two blood components, any amplification seen using the C. albicans-specific assay would reflect cell-associated DNA from spiked C. albicans cells. Conversely, any amplification using the A. fumigatus-specific assay would reflect only cell-free DNA from the spiked genomic DNA of A. fumigatus. A greater proportion of fungal cell-associated DNA was found in the blood pellet fraction than in the plasma. The centrifugation used to separate the whole blood into its two fractions resulted in the denser, intact blastoconidia of C. albicans pelleting to the bottom of the tube with other cellular components of the whole blood. Conversely, fungal cell-free DNA was found in greater quantity in plasma than in the blood pellet. Based upon our in vitro kinetic data and the results demonstrating a partitioning of cell-free DNA into plasma, we hypothesized that plasma would be a useful source for detection of DNA from Candida albicans in a nonneutropenic rabbit model of disseminated candidiasis.
Samples of whole blood obtained from nonneutropenic rabbits with experimental disseminated candidiasis were collected daily and processed throughout the time course of the experiment. Due to the inhibitory effect of heparin on PCR (30), it was important to design our experiments such that the collection of all blood samples was performed in tubes with K2-EDTA as the anticoagulant. Increasing counts of CFU/ml of daily blood pellet cultures were clear indicators that infection had been well established and reflected the progression of disease from day 1 through day 8 of the experiment. Fungal cell-free DNA was found in the plasma fraction as early as day 1 postinoculation, with circulating fungal DNA levels increasing as progression of disease occurred. Unlike the in vitro models, the in vivo experiments were not capacity-limited systems. Therefore, blood cultures showed increased counts of CFU/ml over the time course of the experiment. Parallel levels of cell-free DNA in the plasma reflected the increased concentration of organisms found in the blood pellet. Additionally, the in vitro experiments showed the effect of H-MNCs on the DNA release of C. albicans. In vivo, there are many more host defense factors (e.g., neutrophils) that may play a role in releasing additional C. albicans DNA. Corresponding plasma cultures for circulating C. albicans remained very low to nonexistent, confirming that the fungal DNA detected in plasma was cell-free DNA. These findings are in agreement with previous studies which showed that dead or dying cells have been considered the source of extracellular DNA (4).
Once the DNA had been released from the cell, the issue of DNA stability in the extracellular environment needed to be ascertained. Our studies showed that C. albicans cell-free DNA remained stable in water and rabbit and human plasma over 72 h at various temperatures (room temperature, 4°C, and –30°C). These findings are in agreement with previous studies showing the stability of spiked A. fumigatus DNA in blood samples up to 72 h (11).
Although there have been studies that apply PCR assays to detection of deeply invasive candidiasis (2, 5, 12, 24-27), this to our knowledge is the first report that applies qPCR to investigate the kinetics of DNA release from C. albicans into the extracellular environment and, in addition, describes the role that H-MNCs play in Candida albicans DNA kinetics by increasing the release of fungal cell-free DNA. Further understanding of DNA kinetics of pathogens and the mechanisms by which DNA is released, especially those causing bloodstream infections, should improve strategies in designing and implementing molecular-based diagnostics for fungal pathogens.
ACKNOWLEDGMENTS
We thank Heidi A. Murray and Christine Mya-San for their assistance in conducting laboratory animal studies.
This research was supported by the Intramural Research Program of the NIH and National Cancer Institute.
REFERENCES
Benjamin, D. K., Jr., C. Poole, W. J. Steinbach, J. L. Rowen, and T. J. Walsh. 2003. Neonatal candidemia and end-organ damage: a critical appraisal of the literature using meta-analytic techniques. Pediatrics 112:634-640.
Bu, R., R. K. Sathiapalan, M. M. Ibrahim, I. Al-Mohsen, E. Almodavar, M. I. Gutierrez, and K. Bhatia. 2005. Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus. J. Med. Microbiol. 54:243-248.
Buchman, T. G., M. Rossier, W. G. Merz, and P. Charache. 1990. Detection of surgical pathogens by in vitro DNA amplification. Part I: rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene. Surgery 108:338-346.
Choi, J. J., C. F. Reich III, and D. S. Pisetsky. 2004. Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro. Scand. J. Immunol. 60:159-166.
Chryssanthou, E., B. Andersson, B. Petrini, S. Lofdahl, and J. Tollemar. 1994. Detection of Candida albicans DNA in serum by polymerase chain reaction. Scand. J. Infect. Dis. 26:479-485.
Ellepola, A. N., and C. J. Morrison. 2005. Laboratory diagnosis of invasive candidiasis. J. Microbiol. 43:65-84.
Fleischmann, J., and D. H. Howard. 1988. The DNA content of nongerminative variants of Candida albicans. Nucleic Acids Res. 16:765.
Ghannoum, M. A. 2001. Candida: a causative agent of an emerging infection. J. Investig. Dermatol. Symp. Proc. 6:188-196.
Gudlaugsson, O., S. Gillespie, K. Lee, J. Vande Berg, J. Hu, S. Messer, L. Herwaldt, M. Pfaller, and D. Diekema. 2003. Attributable mortality of nosocomial candidemia, revisited. Clin. Infect. Dis. 37:1172-1177.
Hajjeh, R. A., A. N. Sofair, L. H. Harrison, G. M. Lyon, B. A. Arthington-Skaggs, S. A. Mirza, M. Phelan, J. Morgan, W. Lee-Yang, M. A. Ciblak, L. E. Benjamin, L. T. Sanza, S. Huie, S. F. Yeo, M. E. Brandt, and D. W. Warnock. 2004. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J. Clin. Microbiol. 42:1519-1527.
Hebart, H., J. Loffler, C. Meisner, F. Serey, D. Schmidt, A. Bohme, H. Martin, A. Engel, D. Bunje, W. V. Kern, U. Schumacher, L. Kanz, and H. Einsele. 2000. Early detection of aspergillus infection after allogeneic stem cell transplantation by polymerase chain reaction screening. J. Infect. Dis. 181:1713-1719.
Hopfer, R. L., P. Walden, S. Setterquist, and W. E. Highsmith. 1993. Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis. J. Med. Vet. Mycol. 31:65-75.
Kim, H. S., E. H. Choi, J. Khan, E. Roilides, A. Francesconi, M. Kasai, T. Sein, R. L. Schaufele, K. Sakurai, C. G. Son, B. T. Greer, S. Chanock, C. A. Lyman, and T. J. Walsh. 2005. Expression of genes encoding innate host defense molecules in normal human monocytes in response to Candida albicans. Infect. Immun. 73:3714-3724.
Maaroufi, Y., N. Ahariz, M. Husson, and F. Crokaert. 2004. Comparison of different methods of isolation of DNA of commonly encountered Candida species and its quantitation by using a real-time PCR-based assay. J. Clin. Microbiol. 42:3159-3163.
Miller, L. G., R. A. Hajjeh, and J. E. Edwards, Jr. 2001. Estimating the cost of nosocomial candidemia in the United States. Clin. Infect. Dis. 32:1110.
National Research Council. 1996. Guide for the care and use of laboratory animals. National Academy Press, Washington, D.C.
O'Sullivan, C. E., M. Kasai, A. Francesconi, V. Petraitis, R. Petraitiene, A. M. Kelaher, A. A. Sarafandi, and T. J. Walsh. 2003. Development and validation of a quantitative real-time PCR assay using fluorescence resonance energy transfer technology for detection of Aspergillus fumigatus in experimental invasive pulmonary aspergillosis. J. Clin. Microbiol. 41:5676-5682.
Pappas, P. G., J. H. Rex, J. Lee, R. J. Hamill, R. A. Larsen, W. Powderly, C. A. Kauffman, N. Hyslop, J. E. Mangino, S. Chapman, H. W. Horowitz, J. E. Edwards, and W. E. Dismukes. 2003. A prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients. Clin. Infect. Dis. 37:634-643.
Petraitis, V., R. Petraitiene, A. H. Groll, K. Roussillon, M. Hemmings, C. A. Lyman, T. Sein, J. Bacher, I. Bekersky, and T. J. Walsh. 2002. Comparative antifungal activities and plasma pharmacokinetics of micafungin (FK463) against disseminated candidiasis and invasive pulmonary aspergillosis in persistently neutropenic rabbits. Antimicrob. Agents Chemother. 46:1857-1869.
Pfaller, M. A., and D. J. Diekema. 2004. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42:4419-4431.
Reiss, E., T. Obayashi, K. Orle, M. Yoshida, and R. M. Zancope-Oliveira. 2000. Non-culture based diagnostic tests for mycotic infections. Med. Mycol. 38(Suppl. 1):147-159.
Riggsby, W. S., L. J. Torres-Bauza, J. W. Wills, and T. M. Townes. 1982. DNA content, kinetic complexity, and the ploidy question in Candida albicans. Mol. Cell. Biol. 2:853-862.
Roilides, E., E. Farmaki, J. Evdoridou, A. Francesconi, M. Kasai, J. Filioti, M. Tsivitanidou, D. Sofianou, G. Kremenopoulos, and T. J. Walsh. 2003. Candida tropicalis in a neonatal intensive care unit: epidemiologic and molecular analysis of an outbreak of infection with an uncommon neonatal pathogen. J. Clin. Microbiol. 41:735-741.
Sakai, T., K. Ikegami, E. Yoshinaga, R. Uesugi-Hayakawa, and A. Wakizaka. 2000. Rapid, sensitive and simple detection of candida deep mycosis by amplification of 18S ribosomal RNA gene; comparison with assay of serum beta-D-glucan level in clinical samples. Tohoku J. Exp. Med. 190:119-128.
Skladny, H., D. Buchheidt, C. Baust, F. Krieg-Schneider, W. Seifarth, C. Leib-Msch, and R. Hehlmann. 1999. Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR. J. Clin. Microbiol. 37:3865-3871.
Trama, J. P., E. Mordechai, and M. E. Adelson. 2005. Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing. Mol. Cell. Probes 19:145-152.
Wahyuningsih, R., H.-J. Freisleben, H.-G. Sonntag, and P. Schnitzler. 2000. Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis. J. Clin. Microbiol. 38:3016-3021.
Walsh, T. J., J. Bacher, and P. A. Pizzo. 1988. Chronic silastic central venous catheterization for induction, maintenance and support of persistent granulocytopenia in rabbits. Lab. Anim. Sci. 38:467-471.
Williamson, E. C., and J. P. Leeming. 1999. Molecular approaches for the diagnosis and epidemiological investigation of Aspergillus infection. Mycoses 42(Suppl. 2):7-10.
Woo, P. C., S. K. Lo, K. Y. Yuen, J. S. Peiris, H. Siau, E. K. Chiu, R. H. Liang, and T. K. Chan. 1997. Detection of CMV DNA in bone marrow transplant recipients: plasma versus leucocyte polymerase chain reaction. J. Clin. Pathol. 50:231-235.(Miki Kasai, Andrea France)