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Species identification of medically important fungi by use of real-time LightCycler PCR

¹Laboratory for Mycopathogen, Chlamydia and Mycoplasma, Division of Laboratory Research and Development, Center for Disease Control, Taipei, Taiwan ²Division of Clinical Research, National Health Research Institutes, Taipei, Taiwan ³Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan

Correspondence Shu-Ying Li syl{at}cdc.gov.tw

Received April 25, 2003
Accepted September 8, 2003

Invasive fungal infection has become a major cause of morbidity and mortality in immunocompromised patients. Rapid identification of pathogenic fungi to species level is critical for disease treatment. A real-time LightCycler assay aiming at rapid detection and species identification of pathogenic fungi from clinical isolates was developed. Template DNAs of different species were amplified and detected in real time by employing SYBR Green fluorescent dye. The target sequences for species-level detection were located between the 18S and 28S rDNA. Seven fungal species encountered frequently in the clinical setting, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida guilliermondii and Cryptococcus neoformans, could be discriminated by species-specific primers and confirmed by melting-curve analyses. The range of linearity was from 1 ng to 1 pg (µl^-1 water) and the sensitivity was 1 pg fungal DNA µl^-1. Identification by this real-time PCR method matched biochemical identification for all 58 clinical strains. Therefore, the method is simple, rapid and sensitive enough for detection and identification of several fungal species.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Invasive fungal infections have become major causes of morbidity and mortality among immunocompromised patients (Dasbach et al., 2000; Ellis et al., 2001), such as some neutropenic patients with haematological malignancies and recipients of allogenic bone marrow transplants (Denning, 1998) as well as individuals with AIDS (Mitchell & Perfect, 1995). Candida species are now the fourth most frequent cause of nosocomial blood-stream infections in critically ill patients in the United States. At a teaching hospital in Taiwan, yeast infection was identified as the leading cause of nosocomial infection (Chen et al., 1997). The increasing prevalence of yeast infections highlights the need for simple and rapid methods to identify clinically important fungi in a microbiological laboratory.

Each Candida species has a different degree of susceptibility to common antifungal agents. For instance, Candida krusei is innately resistant and Candida glabrata, Candida guilliermondii and Candida dubliniensis are less susceptible to fluconazole than other Candida species (Orozco et al., 1998; Piemonte et al., 1996). Emergence of secondary resistance in Candida lusitaniae to amphotericin B has also been observed and monitored closely (Pfaller et al., 2003). Current recommendations suggest that invasive fungal infections, such as candidiasis and aspergillosis, should be treated empirically, because the current diagnoses are difficult and time-consuming (Rex et al., 2000; Stevens et al., 2000). However, there is great concern that such practice would result in the emergence of resistant fungal pathogens.

As more and more alternative antifungal agents with various spectra of activities are developed and become available, treatment according to accurate diagnosis has become even more important. Therefore, rapid species identification will be more critical for effective disease therapy and control (Polak, 2003). Conventional diagnostic tests, such as blood culture and biochemical tests, which lack sufficient sensitivity and specificity for early diagnosis of invasive fungal infections, may often require 2 or more days and may be inaccurate (Espinel-Ingroff et al., 1998; Goodwin et al., 1992; Hazen, 1995). Serological tests have certain limitations, e.g. antibody response may be lacking or varied, since the patients most at risk of fungal infections are often immunosuppressed. As for antigen tests, there are no widely accepted Candida antigen tests and most cannot differentiate among Candida species. Other tests, such as those for cryptococcal, aspergillus and histoplasmal antigen, are often hampered by low serum antigen concentrations (Morrison & Lindsley, 2002; Yeo & Wong, 2002).

Therefore, diagnostic assays based on in vitro amplification and detection of fungal DNA have been developed, among which PCR methods are particularly promising because of their high specificity and sensitivity. A number of studies have described restriction fragment length polymorphism, PCR amplification and hybridization with species-specific probes, amplicon size differences (Chen et al., 2001; Fujita et al., 2001; Henry et al., 2000) or other methods to identify unique DNA sequences (Hopfer et al., 1993; Kappe et al., 1998; Martin et al., 2000; Turenne et al., 1999). Although these published PCR methods are quite useful for identification of fungal species, they still require a minimum of several hours for DNA amplification and visualization. More recently, real-time PCR techniques have been developed for the detection of fungal pathogens such as Candida species, Cryptococcus neoformans and Aspergillus species. All these assays demonstrate sensitivities better or at least comparable to previously described PCR methods. Real-time PCR assays dramatically decrease the risk of false-positive results, because the PCR and detection systems are coupled and conducted in a closed system and no laborious post-PCR analyses are required. Various real-time PCR platforms have been developed. The signal to be analysed can be generated by double-stranded DNA-specific dyes, such as SYBR Green, or by sequence-specific fluorescence energy transfer probes. A couple of exonuclease-based TaqMan PCR assays, capable of rapid identification and speciation of six Candida species (Guiver et al., 2001) or Aspergillus fumigatus (Costa et al., 2002; Kami et al., 2001), have been described. DNA detection methods for Candida albicans, A. fumigatus and Cryptococcus neoformans using the LightCycler have also been reported (Bialek et al., 2002; Loeffler et al., 2000; Spiess et al., 2003). White et al. (2003) described real-time and high-sensitivity detection of seven Candida species using the LightCycler system; however, they were not able to speciate them.

In this paper, we describe a simple real-time PCR assay with the LightCycler system employing species-specific primers and SYBR Green fluorescent dye for detection and species identification of fungal strains. This assay offers the advantage that the conventional PCR can be easily adapted to real-time format without the need for complicated probe design. The assay has been shown to be amply specific and sensitive and to facilitate the detection procedure significantly.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Fungal strains.

Eight reference strains and 50 clinical strains of Candida albicans (n = 16), Candida glabrata (n = 9), Candida guilliermondii (n = 8), Candida krusei (n = 5), Candida parapsilosis (n = 8), Candida tropicalis (n = 10) and Cryptococcus neoformans (n = 2) were used in this study (Table 1). Taiwan Surveillance of Antimicrobial Resistance of Yeasts (Lo et al., 2001) collected clinical strains isolated from different body sites and from 22 hospitals located in different geographical locations in Taiwan from 15 April to 15 June 1999. Identification of fungal strains was done by germ-tube assay followed by VITEK yeast biochemical card and API-32C systems. Fungi were cultured on Sabouraud dextrose agar (SDA) plates for 72 h at 37 °C.

DNA extraction.

DNA was extracted by using the PUREGENE DNA purification kit (Gentra). Briefly, two loops of fungal biomass from a 2- to 3-day-old culture on SDA were transferred to a 5-ml sterile tube with parafilm seals and re-suspended in 2 ml PBS containing 10–15 µl lyticase and the mixture was incubated overnight at 37 °C. The samples were centrifuged at 13 000 g for 3 min and the supernatant was removed. Cell lysis solution (2 ml) was added to the cell pellet and gently pipetted up and down to lyse the cells. Next, 1 ml protein precipitation solution was added to the cell lysate and vortexed vigorously at high speed for 20 s and then centrifuged at 13 000 g for 10 min. The supernatant was mixed with 100 % isopropanol to precipitate DNA. The DNA was washed with 70 % ethanol, air-dried and dissolved in 50 µl DNA hydration solution. DNA concentrations were measured with a spectrophotometer (A₂₆₀) and DNA samples were stored at -80 °C until used.

LightCycler-based PCR.

The LightCycler PCR and detection system (Roche Diagnostics) was used for amplification and quantification. PCR was performed in glass capillaries and cycling was achieved by alternating heated air and air of ambient temperature, which ensures rapid equilibration between the air and the reaction components due to the high surface-to-volume ratio of the capillaries. The locations and sequences of the species-specific primers (CALB, CGL, CPA, CTR, CGU, CKRU and CN) are shown in Fig. 1 and Table 2 (Lindsley et al., 2001; Luo & Mitchell, 2002). For amplicon detection, the LightCycler FastStart DNA Master SYBR Green kit was used as described by the manufacturer. The PCR mixture (20 µl) contained Taq polymerase, 1x LightCycler reaction buffer, 3 mM magnesium chloride and 0.5 µM primers. Template DNA was added at a final concentration of 1 ng per 20 µl reaction mixture. Samples were run in parallel by performing 35 cycles of repeated denaturation (5 s at 95 °C), annealing (5 s at 58 °C) and chain extension (25 s at 72 °C). This step was followed by a melting-curve analysis from 60 to 95 °C and, afterwards, cooling to 40 °C. The PCR was completed within 45 min. The PCR process was then monitored by fluorescence quantification of the DNA-binding dye SYBR Green 1 dye for the detection of double-stranded DNA.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Specific primer sites for the real-time PCR.

Agarose gel electrophoresis.

Gel electrophoresis was conducted in TBE buffer (0.1 M Tris/HCl, 0.09 M boric acid, 1 mM EDTA, pH 8.4) at 100 V cm^-1 for 50 min in gels composed of 2.0 % (w/v) agarose (BioWhittaker) and stained in 0.5 µg ethidium bromide ml^-1 for 15 min followed by washing for 15 min with distilled water.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The increasing frequency of invasive fungal infections and the high mortality associated with disseminated fungal diseases have underscored the importance of rapid detection of pathogenic fungi. Prompt detection and accurate speciation may help to improve fungal disease management as a whole and lead to more rational use of antifungals. Traditional identification methods based on phenotypic features are often time-consuming and depend largely on the skill and experience of the technician. Therefore, we evaluated the feasibility of LightCycler PCR amplification of the rDNA region followed by melting-curve analysis for identification of clinically important yeasts.

The specificity of LightCycler amplification was tested by annealing the Candida albicans-specific primers CALB1 and CALB2 with DNA extracted from Candida albicans ATCC 14053. The detection limit was about 1 pg µl^-1 (Fig. 2). Similar results were observed with DNA of other species, i.e. the specific primers for each fungal species react only with DNA from the homologous fungal species (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Sensitivity of the Candida albicans ATCC 14053 DNA-specific primers CALB1 and CALB2. Characteristic dose-dependent melting peaks were observed in the presence of DNA from Candida albicans ATCC 14053 [1 ng () to 1 pg () µl-1].

The range of linearity was tested by amplification of 10-fold serial dilutions of purified Candida albicans ATCC 14053 DNA with fluorescence plotted against number of cycles. Linearity was achieved over 4 logs of input fungal DNA amount, from 1 ng to 1 pg µl^-1 (Fig. 3).

View larger version (24K): [in this window] [in a new window]   Fig. 3. (a) Quantification of serially diluted Candida albicans ATCC 14053 DNA [1 ng to 1 pg µl-1] with CALB1 and CALB2 primers using the LightCycler software. (b) Log DNA concentration (fg µl-1) against fluorescence. Slope (-3.924), y-intercept (40.31), mean squared error (0.100) and regression coefficient (-1.00) of the standard curve were calculated.

Compared with bacteraemia or viraemia, fungaemia has much lower pathogen load. Therefore, high-sensitivity detection is pivotal for timely management of fungal diseases. The sensitivity of the assays was evaluated by amplifying DNAs of six Candida species and Cryptococcus neoformans by their respective primers with the LightCycler, and the detection limit of this method was about 1 pg DNA µl^-1. Serially diluted samples of reference strains ranging from 1 ng to 1 pg DNA µl^-1 showed a single band with the specific primers in all cases (Fig. 4).

View larger version (39K): [in this window] [in a new window]   Fig. 4. Agarose electrophoretic gel of LightCycler-amplified DNA from Candida albicans ATCC 14053, showing a single, specific band of 275 bp. Lanes: M; marker; 1, 1 ng µl-1; 2, 100 pg µl-1; 3, 10 pg µl-1; 4, 1 pg µl-1; 5, 100 fg µl-1; 6, 10 fg µl-1; 7, 1 fg µl-1; N, negative control (distilled water).

In order to determine the utility of the LightCycler species-specific amplification method for accurate identification of fungal species, analyses of clinical strains were conducted. The identification results from our real-time PCR method matched completely with biochemical identification results for all 58 tested samples. Characteristic peak T_m for species-specific primers with their respective fungal species were obtained by melting-curve analyses (Table 3).

Targets for species-level detection of fungal pathogens include the 18S rDNA, mitochondrial DNA (Yamada et al., 2002), the internal transcribed spacer (ITS) regions and many other genes (Kanbe et al., 2002). rDNA offers distinct advantages over other molecular targets because of greatly increased sensitivity due to the existence of approximately 100 copies per genome. Many Candida species can be differentiated by analysis of fragment length variation in ITS1 (Chen et al., 2001) or ITS2 (De Baere et al., 2002). The LightCycler system offers another advantage of analysis of the melting temperature of amplicons. The melting temperature of the amplicon is dependent on the G+C content, sequence length and compositional variation in the nucleotide bases. Each fungal species has a characteristic T_m, which helps further to confirm its identity.

In conclusion, the real-time LightCycler PCR assay combines rapid amplification of DNA with real-time species determination. The routine block cycler PCR protocol can be easily transferred and adapted to the real-time protocol. This method is simple, rapid and sensitive and can therefore streamline the flow of diagnostic laboratory work.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We would like to express our sincere appreciation to all 22 participating hospitals for providing strains and information related to these strains. They are Chang Gung Memorial Hospital at Linkou, Chang Gung Memorial Hospital at Keelung, Hsin-Chu Hospital, Lo-Tung Poh Ai Hospital, St Mary Hospital, Taipei Municipal Yang-Ming Hospital, Taipei Municipal Zen Ai Hospital, Tao-Yuan General Hospital, Taiwan Adventist Hospital, Koo Foundation Sun Yat-sen cancer center, Tri Service General Hospital, Kuan-Tien General Hospital, Veterans General Hospital-Taichung, Zen Ai General Hospital, Chi Mei Hospital, Kaohsiung Medical College Chung-Ho Memorial Hospital, Kaohsiung Military Hospital, Tainan Municipal Hospital, Veterans General Hospital – Kaohsiung, Buddhist Tzu-Chi General Hospital in Hua-Lien and Mackay Memorial Hospital Taitung Branch.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES