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Rapid identification of medically important Candida to species level by polymerase chain reaction and single-strand conformational polymorphism
Diagnostic Microbiology and Infectious Disease 38 (2000) 95–99
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Rapid identification of medically important Candida to species level by polymerase chain reaction and single-strand conformational polymorphism Mamie Hui*, Margaret Ip, Paul K.S. Chan, Miu Ling Chin, Augustine F.B. Cheng Department of Microbiology, the Chinese University of Hong Kong, Hong Kong Received 18 April 2000; accepted 27 June 2000
Abstract Invasive fungal disease has taken a great toll on immunocompromised patients. With the emergence of fluconazole and amphotericin B resistance, the rapid identification of fungi to species level is of clinical relevance in guiding appropriate antifungal therapy. Among these opportunistic fungi, Candida species are the most commonly encountered. We had developed a molecular method utilizing single-strand conformational polymorphism (SSCP) to delineate different patterns on a 260-bp amplicon from the 28S rRNA gene from six medically important Candida species. The SSCP banding patterns obtained from a total of 52 isolates were sufficiently unique to allow distinction between the species, thus indicated a high level of specificity. This method of PCR-SSCP can provide a simple and specific method for the rapid identification of medically important Candida to species level. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Invasive fungal infection has emerged as an important infective complication in immunocompromised patients and is associated with high morbidity and mortality (Jarvis 1995; Verduyn et al., 1999). Clinical diagnosis has often been difficult, and the empirical use of antifungal agents are expensive and not without toxicity (Blumberg & Reboli, 1996; Phillips et al., 1997; Viscoli et al., 1996). Laboratory diagnosis by conventional culture methods (Warren & Hazen, 1995) are insensitive, and even when a positive culture is obtained, identification of the fungal pathogen is time consuming and difficult. Among the vast range of opportunistic fungi, Candida species are the most commonly encountered. (Farina et al. 1999; Jarvis, 1995; Richardson & Kokki, 1998; Warnock, 1995). In particular, Candida glabrata, Candida krusei, Candida lusitaniae and Candida guilliermondii are of special concern because of their intrinsic resistance to fluconazole or amphotericin B (Abi-Said et al., 1997; Girmenia & Martino, 1998; Pfaller et al., 1999; Pfaller et al., 2000). A rapid and specific identification method is a requisite to * Corresponding author. Tel.: ⫹852-2632-3333; fax: ⫹852-26473227. E-mail address: [email protected] (M. Hui).
guide the choice of antifungal therapy. Conventional methods for identification by carbohydrate assimilation tests (API 20C/32C), examination of microscopic and macroscopic morphology, or CHROMagar are expensive and technically demanding. Therefore, molecular methods had been investigated. Different methods had been explored by various investigators: panfungal PCR assay (Holmes et al., 1994; Jordan, 1994; Makimura et al., 1994; Miyakawa et al., 1994; Niesters et al., 1993; van Burik et al., 1998), nucleic acid probes (Einsele et al., 1997; Sandhu et al., 1995; Shin et al., 1999), restriction enzymes analysis (Maiwald et al., 1993; Morace et al., 1997; Williams et al., 1995), enzyme immunosorbent assay (Burnie et al., 1997; Fujita et al., 1995; Shin et al., 1997), and PCR-SSCP (Walsh et al., 1995). Panfungal PCR assay had its merit of being rapid and sensitive, especially when used in combination with Southern blotting. Nevertheless, it lacked specificity that was needed in order to distinguish specific species of innate antifungal resistance. Investigators had tried to improve the specificity by using numerous nucleic acid probes and restriction enzymes analysis. The results were reported to be promising, yet these methods required a large panel of enzymes or probes, thus became time consuming and obviated its adoption in the clinical laboratory. Enzyme immunoassay was a simple method with the potential to be
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automated, but was hindered by its lack of sensitivity and specificity. The molecular method of polymerase chain reaction and single strand conformational polymorphism (PCR-SSCP) is a powerful tool for the detection of subtle variations in sequences. It had been employed in numerous aspects of infectious diseases such as the elucidation of rpoB gene mutation in rifampicin resistance of Mycobacterium tuberculosis (Telenti et al., 1993) and the rapid diagnosis of bacteremia in blood cultures (Turenne et al., 2000). PCRSSCP has the advantages of being both sensitive and specific, simple, and without the need for a large panel of restriction enzymes and probes. This method had been utilized in the analysis of different Aspergillus species (Walsh et al., 1995) based on the 197-bp amplicon within the 18S rRNA. The objective of this study was to develop a molecular method that utilized PCR-SSCP for the identification of medically important Candida to the species level.
2. Materials and methods 2.1. Isolates A total of 52 Candida isolates were used in the study. These included six reference ATCC strains and 46 clinical isolates. The ATCC strains were: Candida albicans (ATCC 24433), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258, 14243), Candida parapsilosis (ATCC 22019) and Candida tropicalis (ATCC 750). The clinical isolates were obtained from clinical blood culture isolates from the Prince of Wales Hospital in Hong Kong. They were: eight Candida tropicalis, three Candida glabrata, 16 Candida parapsilosis, one Candida guilliermondii, two Candida lusitaniae and 16 Candida albicans. In addition, two standard strains of Cryptococcus neoformans (ATCC 90112, 90113) and one clinical strain of Trichosporon beigelii were also included for comparison. The identity of the isolates was confirmed by a combination of microscopic appearance on slide cultures on cornmeal agar, carbohydrate assimilation patterns on API 32C strips (bioMerieux, Missouri, USA) and CHROMagar Candida plates (CHROMagar Company, Paris, France). Yeasts were stored on slants of Sabouraud agar (Oxoid, UK) until use.
subsequently resuspended in 50l of distilled water. 0.5l of this DNA solution was utilized as templates in the PCR reactions. 2.3. PCR PCR was performed by using the universal fungal primers binding to the conserved regions within the 28S rRNA (Sandhu et al., 1995). The primers used were: 5⬘-GTGAAATTGTTGAAAGGGAA-3⬘ and 5⬘-GACTCCTTGGTCCGTGTT-3⬘ (Genset, Singapore). A final PCR reaction volume of 100l contained 10l of 10X reaction buffer, 5U Taq polymerase (Phamacia, Uppsala, Sweden), 10mM of each deoxynucleoside triphosphate (Pharmacia), 30M of each primer and 1ng DNA template. Thirty cycles of amplification were performed in a thermalcycler (9700 series, Perkin-Elmer, Emeryville, Calif.) with an initial denaturation step at 94°C for 5 minutes, annealing step at 55°C for 2 minutes, extension step at 72°C for 3 minutes, and a final extension at 72°C for 5 minutes. Throughout the PCR procedure, precautions were taken to avoid contamination of the PCR products (Kwok & Higuchi, 1989). Negative controls were also included in each test run, with reaction mixture omitting either the primers or the DNA templates. The specificity of the PCR was also performed by using templates prepared from with the following standard strains of bacteria: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Enterococcus fecalis (ATCC 700323), Enterococcus cloacacea (ATCC 19433), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 27853) and Acinetobacter baumanii (ATCC 19606). 2.4. SSCP PCR products were mixed with equal volumes of a denaturing solution (95% formamide, 0.05% xylene cyanol solution, 0.01% bromophenol blue) and heated at 95°C for 5 minutes. The mixture was then immediately placed on ice. 4l of this mixture was loaded onto the 12% ExcelGel (Pharmacia) and subjected to electrophoresis under 600V and a constant gel temperature of 4°C or 15°C for 90 minutes. The SSCP patterns were visualized by silver staining.
2.2. Extraction of DNA
3. Results
A single colony of yeast cells was suspended in 200l lysis buffer (100mM Tris-HCl, pH 7.5, sodium dodecyl sulphate solution 0.5% w/v, 30mM EDTA). The mixture was incubated at 100°C for 15 minutes, 100l of 2.5M potassium acetate was then added and mixed. After being stored on ice for 60 minutes, it was centrifuged at 12000rpm for 5 minutes. The supernatant was transferred to a new tube. DNA was precipitated with an equal volume of isopropanol, washed with 0.5ml of 70% ethanol, air-dried and
PCR amplicons of 260-bp were obtained from all ATCC strains of yeasts and clinical strains of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida lusitaniae, Cryptococcus neoformans, and Trichosporon beigelii. No false amplification was observed with all the eight ATCC bacterial strains nor the reagent controls, indicating the specificity of the PCR assay. The SSCP banding patterns were indistinguishable when
M. Hui et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 95–99
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Fig. 1. SSCP patterns of the 260-bp amplicons of different yeast strains at 15°C. Lane 1: HindIII/174 marker; Lane 2: Trichosporon beigelii; Lanes 3– 4: Candida lusitaniae; Lane 5: Candida guilliermondii; Lane 6: Cryptococcus neoformans (ATCC 90112); Lane 7: Cryptococcus neoformans (ATCC 90113); Lane 8: Candida krusei (ATCC 6258); Lane 9: Candida krusei (ATCC 14243); Lanes 10 –11: Candida tropicalis; Lane 12: Candida tropicalis (ATCC 750); Lanes 13–14: Candida glabrata; Lane 15: Candida glabrata (ATCC 90030); Lanes 16 –17: Candida albicans; Lane 18: Candida albicans (ATCC 24433); Lanes 19 –20: Candida parapsilosis; Lane 21: Candida parapsilosis (ATCC 22019). Lanes 2– 4, 10 –11, 13–14, 16 –17, 19 –20 were clinical isolates.
the experiment was run at 4°C. The banding patterns became unique at 15°C to allow distinction between the eight tested yeasts: Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida lusitaniae, Cryptococcus neoformans, and Trichosporon beigelii (Fig. 1). For the following Candida species: Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida lusitaniae and Candida parapsilosis, the SSCP banding patterns obtained among the same species were identical, therefore allowing identification of medically important Candida to the species level.
4. Discussion Conventional methods for identification of fungi have always been difficult, time consuming and technically demanding. In this study, we demonstrated that the 28S-rRNA based PCR-SSCP method was capable of detecting and rapidly identifying a wide range of medically important
Candida species. The advantages of this molecular method were simple to perform, specific and practical. Its application can also be extended with modifications for use in diagnosis of fungemia from patients’ blood cultures. A simple method of DNA extraction was employed. Unlike previous investigators, who used enzymes to produce spheroplasts (Walsh et al., 1995), our method involved the use of chemical lysis buffer with heating; this greatly simplified the procedures and reduced the time required for the extraction of DNA. The primers chosen was based on the highly conserved regions within the 28S rRNA. We were able to amplify the desired sequences without any false amplification from other bacterial isolates. Contamination of PCR reactions was also not observed. Throughout the experiment, the recommendations made by Kwok and Higuchi were strictly followed to minimize the potential of contamination, not only from carry-over of PCR products, but also from environmental saprophytic fungi. At 4°C, there was no distinguishable SSCP pattern among the eight species of yeasts. However, when the
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temperature was raised to 15°C, unique banding patterns were obtained among the different organisms. Walsh et al., had also observed similar phenomenon, in their experiment, the Aspergillus species banding were the same at 4°C, but the banding patterns became unique when the temperature was raised to room temperature. Since the results of SSCP is dependent on the conditions used in the experiment (Nataraj et al., 1999), thus such findings were expected and the temperature of the experiment needs to be optimized. The number of isolates for Candida krusei (n ⫽ 2), Candida guilliermondii (n ⫽ 1) and Candida lusitaniae (n ⫽ 2) was limited, as compared with the other Candida species. A further study to include a larger number of these isolates would be needed to further ascertain the discriminatory power of the technique. A number of other Candida species were not included in the present study, e.g., Candida kefry. However, the above panel of fungi investigated was the majority of Candida isolates seen in our clinical laboratory (unpublished data). In the event of invasive fungemia, the time required to obtain a specific organism identity is critical to patient management. In this study, by running each of the species as controls for comparison, an unknown Candida species can be identified within one working day. This provides an opportunity for the early adjustment of antifungal therapy, when a specific species is identified that possess innate antifungal resistance. Therefore, we described a method of PCR-SSCP that is rapid, simple and specific, which can be easily adopted for use in clinical microbiology laboratory for the identification of medically important Candida.
Acknowledgment This work was supported by a grant from the Chinese University of Hong Kong (2040689).
References Abi-Said, D., Anaissie, E., Uzun, O., Raad, I., Pinzcowski, H., & Vartivarian, S. (1997). The epidemiology of hematogenous candidiasis caused by different Candida species. Clin Infect Dis 24, 1122–1128. Blumberg, E. A., & Reboli, A. C. (1996). Failure of systemic empirical treatment with amphotericin B to prevent candidemia in neutropenic patients with cancer. Clin Infect Dis 22, 462– 6. Burnie, J. P., Golbang, N., & Matthews, R. C. (1997). Semiquantitative polymerase chain reaction enzyme immunoassay for diagnosis of disseminated candidiasis. Eur J Clin Microbiol Infect Dis 16, 346 –350. Einsele, H., Herbert, H., & Roller, G., et al. (1997). Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 35, 1353–1360. Farina, C., Vailati, F., Manisco, A., & Goglio, A. (1999). Fungaemia survey: a 10-year experience in Bergamo, Italy. Mycoses 42, 543– 8. Fujita, S. H., Lasker, B. A., Lott, T. J., Reiss, E., & Morrison, C. J. (1995). Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood. J Clin Microbiol 33, 962–967.
Girmenia, C., & Martino, P. (1998). Fluconazole and the changing epidemiology of candidemia. Clin Infect Dis 27, 234. Holmes, A. R., Cannon, R. D., Shepherd, M. G., & Jenkinson, H. F. (1994). Detection of Candida albicans and other yeasts in blood by PCR J Clin Microbiol 32, 228 –231. Jarvis, W. R. (1995). Epidemiology of nosocomical fungal infections, with emphasis on Candida species. Clin Infect Dis 20, 1526 –1530. Jordan, J. A. (1994). PCR identification of four medically important Candida species by using a single primer pair. J Clin Microbiol 32, 2962–2967. Kwok, S., & Higuchi, R. (1989). Avoiding false positives with PCR. Nature 339, 237–238. Maiwald, M., Kappe, R., & Sonntag, H. G. (1994). Rapid presumptive identification of medically relevant yeasts to the species level by polymerase chain reaction and restriction enzyme analysis. J Med Vet Mycol 32, 115–122. Makimura, K., Murayama, S. Y., & Yamaguchi, H. (1994). Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 40, 358 –364. Miyakawa, Y., Mabuchi, T., & Fukazawa, Y. (1993). New method for detection of Candida albicans in human blood by polymerase chain reaction. J Clin Microbiol 31, 3344 –3347. Morace, G., Sanguinetti, M., Posteraro, B., Lo Cascio, G., & Fadda, G. (1997). Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis. J Clin Microbiol 35, 667– 672. Nataraj, A. J., Olivos-Glander, I., Kusukawa, N., & Highsmith, W. E., Jr. (1999). Single strand conformation polymorphism and heteroduplex analysis of gel-based mutation detection. Electrophoresis 20, 1177– 1185. Niesters, H. G., Goessens, W. H., Meis, J. F., & Quint, W. G. (1993). Rapid, polymerase chain reaction based identification assays for Candida species. J Clin Microbiol 31, 904 –910. Pfaller, M. A., Jones, R. N., Doern, G. V., Fluit, A. C., Verhoef, J., & Sader, H. S., et al. (1999). International surveillance of blood stream infections due to Candida species in the European SENTRY program: species distribution and antifungal susceptibility agents. SENTRY participant group (Europe). Diagn Microbiol Infect Dis 35, 19 –25. Pfaller, M. A., Jones, R. N., Doern, G. V., Sader, H. S., Messer, S. A., & Houston, A., et al. (2000). Bloodstream infections due to Candida species: SENTRY antimicrobial surveillance program in North America and latin america, 1997–1998. Antimicrob Agents Chemother 44, 747–751. Phillips, P., Shafran, S., Garber, G., Rotstein, C., Smaill, F., & Fong, I., et al. (1997). Multicentre randomized trial of fluconazole versus amphotericin B for treatment f candidemia in non-neutropenic patients. Canadian Candidemia study group. Eur J Clin Microbiol Infect Dis 16, 337–345. Richardson, M. D., & Kokki, M. H. (1998). Diagnosis and prevention of fungal infection in the immunocompromized patient. Blood Rev 12, 241–254. Sandhu, G. S., Kline, B. C., Stockman, L., & Roberts, G. D. (1995). Molecular probes for diagnosis of fungal infections. J Clin Microbiol 33, 2913–2919. Shin, J. H., Nolte, F. S., & Morrison, C. J. (1997). Rapid identification of Candida species in a blood cultures by a clinically useful PCR method. J Clin Microbiol 35, 1454 –1459. Shin, J. H., Nolte, F. S., Holloway, B. P., & Morrison, C. J. (1999). Rapid identification of up to three Candida species in a single reaction tube by a 5⬘ exonuclease assay using fluorescent DNA probes. J Clin Microbiol 37, 165–170. Telenti, A., Imboden, P., Marchesi, F., Lowrie, D., Cole, S., & Colston, M. J., et al. (1993). Detection of rifampicin resistance mutations in Mycobacterium tuberculosis. Lancet 341, 647– 650. Turenne, C. Y., Witwicki, E., Hoban, D. J., Karlowsky, J. A., & Kabani, A. M. (2000). Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand-conformation poly-
M. Hui et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 95–99 morphism analysis of the 16S rRNA gene. J Clin Microbiol 38, 513– 520. van Burik, J. A., Myerson, D., Schreckhise, R. W., & Bowden, R. A. (1998). Panfungal PCR assay for detection of fungal infection in human blood specimens. J Clin Microbiol 36, 1169 –1175. Verduyn, L. F. M., Meis, J. F., & Voss, A. (1999). Nosocomial fungal infections: candidemia. Diagn Microbiol Infect Dis 34, 213–220. Viscoli, C., Castagnola, E., Van Lint, M. T., Moroni, C., Garaventa, A., & Rossi, M. R., et al. (1996). Fluconazole versus amphotericin B as empirical antifungal therapy of unexplained fever in granulocytopenic cancer patients: a pragmatic, multicentre, prospective and randomized clinical trial. Eur J Cancer 32A, 814 – 820. Walsh, T. J., Francesconi, A., Kasai, M., & Chanock, S. J. (1995). PCR and single-strand conformational polymorphism for recognition of
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medically important opportunistic fungi. J Clin Microbiol 33, 3216 –3220. Warnock, D. W. (1995). Fungal complications of transplantation: diagnosis, treatment and prevention. J Antimicrob Chemother 36(Suppl B), 73–90. Warren, N. G., & Hazen, K. C. (1995). Candida, Cryptococcus, and other yeasts of medical importance. In P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, R. H. Yolken, (Ed.), Manual of clinical microbiology, 6th ed. (pp. 723–737). Washington, D.C.: American Society of Microbiology. Williams, D. W., Wilson, M. J., Lewis, M. A. O., & Potts, A. J. C. (1995). Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J Clin Microbiol 33, 2476 –2479.
www.elsevier.com/locate/diagmicrobio
Rapid identification of medically important Candida to species level by polymerase chain reaction and single-strand conformational polymorphism Mamie Hui*, Margaret Ip, Paul K.S. Chan, Miu Ling Chin, Augustine F.B. Cheng Department of Microbiology, the Chinese University of Hong Kong, Hong Kong Received 18 April 2000; accepted 27 June 2000
Abstract Invasive fungal disease has taken a great toll on immunocompromised patients. With the emergence of fluconazole and amphotericin B resistance, the rapid identification of fungi to species level is of clinical relevance in guiding appropriate antifungal therapy. Among these opportunistic fungi, Candida species are the most commonly encountered. We had developed a molecular method utilizing single-strand conformational polymorphism (SSCP) to delineate different patterns on a 260-bp amplicon from the 28S rRNA gene from six medically important Candida species. The SSCP banding patterns obtained from a total of 52 isolates were sufficiently unique to allow distinction between the species, thus indicated a high level of specificity. This method of PCR-SSCP can provide a simple and specific method for the rapid identification of medically important Candida to species level. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Invasive fungal infection has emerged as an important infective complication in immunocompromised patients and is associated with high morbidity and mortality (Jarvis 1995; Verduyn et al., 1999). Clinical diagnosis has often been difficult, and the empirical use of antifungal agents are expensive and not without toxicity (Blumberg & Reboli, 1996; Phillips et al., 1997; Viscoli et al., 1996). Laboratory diagnosis by conventional culture methods (Warren & Hazen, 1995) are insensitive, and even when a positive culture is obtained, identification of the fungal pathogen is time consuming and difficult. Among the vast range of opportunistic fungi, Candida species are the most commonly encountered. (Farina et al. 1999; Jarvis, 1995; Richardson & Kokki, 1998; Warnock, 1995). In particular, Candida glabrata, Candida krusei, Candida lusitaniae and Candida guilliermondii are of special concern because of their intrinsic resistance to fluconazole or amphotericin B (Abi-Said et al., 1997; Girmenia & Martino, 1998; Pfaller et al., 1999; Pfaller et al., 2000). A rapid and specific identification method is a requisite to * Corresponding author. Tel.: ⫹852-2632-3333; fax: ⫹852-26473227. E-mail address: [email protected] (M. Hui).
guide the choice of antifungal therapy. Conventional methods for identification by carbohydrate assimilation tests (API 20C/32C), examination of microscopic and macroscopic morphology, or CHROMagar are expensive and technically demanding. Therefore, molecular methods had been investigated. Different methods had been explored by various investigators: panfungal PCR assay (Holmes et al., 1994; Jordan, 1994; Makimura et al., 1994; Miyakawa et al., 1994; Niesters et al., 1993; van Burik et al., 1998), nucleic acid probes (Einsele et al., 1997; Sandhu et al., 1995; Shin et al., 1999), restriction enzymes analysis (Maiwald et al., 1993; Morace et al., 1997; Williams et al., 1995), enzyme immunosorbent assay (Burnie et al., 1997; Fujita et al., 1995; Shin et al., 1997), and PCR-SSCP (Walsh et al., 1995). Panfungal PCR assay had its merit of being rapid and sensitive, especially when used in combination with Southern blotting. Nevertheless, it lacked specificity that was needed in order to distinguish specific species of innate antifungal resistance. Investigators had tried to improve the specificity by using numerous nucleic acid probes and restriction enzymes analysis. The results were reported to be promising, yet these methods required a large panel of enzymes or probes, thus became time consuming and obviated its adoption in the clinical laboratory. Enzyme immunoassay was a simple method with the potential to be
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M. Hui et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 95–99
automated, but was hindered by its lack of sensitivity and specificity. The molecular method of polymerase chain reaction and single strand conformational polymorphism (PCR-SSCP) is a powerful tool for the detection of subtle variations in sequences. It had been employed in numerous aspects of infectious diseases such as the elucidation of rpoB gene mutation in rifampicin resistance of Mycobacterium tuberculosis (Telenti et al., 1993) and the rapid diagnosis of bacteremia in blood cultures (Turenne et al., 2000). PCRSSCP has the advantages of being both sensitive and specific, simple, and without the need for a large panel of restriction enzymes and probes. This method had been utilized in the analysis of different Aspergillus species (Walsh et al., 1995) based on the 197-bp amplicon within the 18S rRNA. The objective of this study was to develop a molecular method that utilized PCR-SSCP for the identification of medically important Candida to the species level.
2. Materials and methods 2.1. Isolates A total of 52 Candida isolates were used in the study. These included six reference ATCC strains and 46 clinical isolates. The ATCC strains were: Candida albicans (ATCC 24433), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258, 14243), Candida parapsilosis (ATCC 22019) and Candida tropicalis (ATCC 750). The clinical isolates were obtained from clinical blood culture isolates from the Prince of Wales Hospital in Hong Kong. They were: eight Candida tropicalis, three Candida glabrata, 16 Candida parapsilosis, one Candida guilliermondii, two Candida lusitaniae and 16 Candida albicans. In addition, two standard strains of Cryptococcus neoformans (ATCC 90112, 90113) and one clinical strain of Trichosporon beigelii were also included for comparison. The identity of the isolates was confirmed by a combination of microscopic appearance on slide cultures on cornmeal agar, carbohydrate assimilation patterns on API 32C strips (bioMerieux, Missouri, USA) and CHROMagar Candida plates (CHROMagar Company, Paris, France). Yeasts were stored on slants of Sabouraud agar (Oxoid, UK) until use.
subsequently resuspended in 50l of distilled water. 0.5l of this DNA solution was utilized as templates in the PCR reactions. 2.3. PCR PCR was performed by using the universal fungal primers binding to the conserved regions within the 28S rRNA (Sandhu et al., 1995). The primers used were: 5⬘-GTGAAATTGTTGAAAGGGAA-3⬘ and 5⬘-GACTCCTTGGTCCGTGTT-3⬘ (Genset, Singapore). A final PCR reaction volume of 100l contained 10l of 10X reaction buffer, 5U Taq polymerase (Phamacia, Uppsala, Sweden), 10mM of each deoxynucleoside triphosphate (Pharmacia), 30M of each primer and 1ng DNA template. Thirty cycles of amplification were performed in a thermalcycler (9700 series, Perkin-Elmer, Emeryville, Calif.) with an initial denaturation step at 94°C for 5 minutes, annealing step at 55°C for 2 minutes, extension step at 72°C for 3 minutes, and a final extension at 72°C for 5 minutes. Throughout the PCR procedure, precautions were taken to avoid contamination of the PCR products (Kwok & Higuchi, 1989). Negative controls were also included in each test run, with reaction mixture omitting either the primers or the DNA templates. The specificity of the PCR was also performed by using templates prepared from with the following standard strains of bacteria: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Enterococcus fecalis (ATCC 700323), Enterococcus cloacacea (ATCC 19433), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 27853) and Acinetobacter baumanii (ATCC 19606). 2.4. SSCP PCR products were mixed with equal volumes of a denaturing solution (95% formamide, 0.05% xylene cyanol solution, 0.01% bromophenol blue) and heated at 95°C for 5 minutes. The mixture was then immediately placed on ice. 4l of this mixture was loaded onto the 12% ExcelGel (Pharmacia) and subjected to electrophoresis under 600V and a constant gel temperature of 4°C or 15°C for 90 minutes. The SSCP patterns were visualized by silver staining.
2.2. Extraction of DNA
3. Results
A single colony of yeast cells was suspended in 200l lysis buffer (100mM Tris-HCl, pH 7.5, sodium dodecyl sulphate solution 0.5% w/v, 30mM EDTA). The mixture was incubated at 100°C for 15 minutes, 100l of 2.5M potassium acetate was then added and mixed. After being stored on ice for 60 minutes, it was centrifuged at 12000rpm for 5 minutes. The supernatant was transferred to a new tube. DNA was precipitated with an equal volume of isopropanol, washed with 0.5ml of 70% ethanol, air-dried and
PCR amplicons of 260-bp were obtained from all ATCC strains of yeasts and clinical strains of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida lusitaniae, Cryptococcus neoformans, and Trichosporon beigelii. No false amplification was observed with all the eight ATCC bacterial strains nor the reagent controls, indicating the specificity of the PCR assay. The SSCP banding patterns were indistinguishable when
M. Hui et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 95–99
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Fig. 1. SSCP patterns of the 260-bp amplicons of different yeast strains at 15°C. Lane 1: HindIII/174 marker; Lane 2: Trichosporon beigelii; Lanes 3– 4: Candida lusitaniae; Lane 5: Candida guilliermondii; Lane 6: Cryptococcus neoformans (ATCC 90112); Lane 7: Cryptococcus neoformans (ATCC 90113); Lane 8: Candida krusei (ATCC 6258); Lane 9: Candida krusei (ATCC 14243); Lanes 10 –11: Candida tropicalis; Lane 12: Candida tropicalis (ATCC 750); Lanes 13–14: Candida glabrata; Lane 15: Candida glabrata (ATCC 90030); Lanes 16 –17: Candida albicans; Lane 18: Candida albicans (ATCC 24433); Lanes 19 –20: Candida parapsilosis; Lane 21: Candida parapsilosis (ATCC 22019). Lanes 2– 4, 10 –11, 13–14, 16 –17, 19 –20 were clinical isolates.
the experiment was run at 4°C. The banding patterns became unique at 15°C to allow distinction between the eight tested yeasts: Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida lusitaniae, Cryptococcus neoformans, and Trichosporon beigelii (Fig. 1). For the following Candida species: Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida lusitaniae and Candida parapsilosis, the SSCP banding patterns obtained among the same species were identical, therefore allowing identification of medically important Candida to the species level.
4. Discussion Conventional methods for identification of fungi have always been difficult, time consuming and technically demanding. In this study, we demonstrated that the 28S-rRNA based PCR-SSCP method was capable of detecting and rapidly identifying a wide range of medically important
Candida species. The advantages of this molecular method were simple to perform, specific and practical. Its application can also be extended with modifications for use in diagnosis of fungemia from patients’ blood cultures. A simple method of DNA extraction was employed. Unlike previous investigators, who used enzymes to produce spheroplasts (Walsh et al., 1995), our method involved the use of chemical lysis buffer with heating; this greatly simplified the procedures and reduced the time required for the extraction of DNA. The primers chosen was based on the highly conserved regions within the 28S rRNA. We were able to amplify the desired sequences without any false amplification from other bacterial isolates. Contamination of PCR reactions was also not observed. Throughout the experiment, the recommendations made by Kwok and Higuchi were strictly followed to minimize the potential of contamination, not only from carry-over of PCR products, but also from environmental saprophytic fungi. At 4°C, there was no distinguishable SSCP pattern among the eight species of yeasts. However, when the
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temperature was raised to 15°C, unique banding patterns were obtained among the different organisms. Walsh et al., had also observed similar phenomenon, in their experiment, the Aspergillus species banding were the same at 4°C, but the banding patterns became unique when the temperature was raised to room temperature. Since the results of SSCP is dependent on the conditions used in the experiment (Nataraj et al., 1999), thus such findings were expected and the temperature of the experiment needs to be optimized. The number of isolates for Candida krusei (n ⫽ 2), Candida guilliermondii (n ⫽ 1) and Candida lusitaniae (n ⫽ 2) was limited, as compared with the other Candida species. A further study to include a larger number of these isolates would be needed to further ascertain the discriminatory power of the technique. A number of other Candida species were not included in the present study, e.g., Candida kefry. However, the above panel of fungi investigated was the majority of Candida isolates seen in our clinical laboratory (unpublished data). In the event of invasive fungemia, the time required to obtain a specific organism identity is critical to patient management. In this study, by running each of the species as controls for comparison, an unknown Candida species can be identified within one working day. This provides an opportunity for the early adjustment of antifungal therapy, when a specific species is identified that possess innate antifungal resistance. Therefore, we described a method of PCR-SSCP that is rapid, simple and specific, which can be easily adopted for use in clinical microbiology laboratory for the identification of medically important Candida.
Acknowledgment This work was supported by a grant from the Chinese University of Hong Kong (2040689).
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