An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature-shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits.

Centre for Cardiovascular Surgery and Transplantation Brno Brno Czech Republic; International Clinical Research Centre St Anne's University Hospital Brno Brno Czech Republic

Centre for Cardiovascular Surgery and Transplantation Brno Brno Czech Republic; Molecular Immunology and Microbiology Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Microbiology Faculty of Medicine Masaryk University and St Anne's University Hospital Brno Brno Czech Republic

Molecular Immunology and Microbiology Central European Institute of Technology Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Wisplinghoff H, Bischoff T, Tallent M, Seifert H, Wenzel R, et al. (2004) Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis: 1093–1093. PubMed

Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20: 133–163. PubMed PMC

Marchetti O, Bille J, Fluckiger U, Eggimann P, Ruef C, et al. (2004) Epidemiology of candidemia in Swiss tertiary care hospitals: Secular trends, 1991–2000. Clin Infect Dis: 311–320. PubMed

Tortorano A, Peman J, Bernhardt H, Klingspor L, Kibbler C, et al. (2004) Epidemiology of candidaemia in Europe: Results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study. Eur J Clin MicrobiolInfect Dis: 317–322. PubMed

Krcmery V, Barnes A (2002) Non-albicans Candida spp. causing fungaemia: pathogenicity and antifungal resistance. J Hosp Infect: 243–260. PubMed

Pfaller MA, Diekema DJ (2002) Role of sentinel surveillance of candidemia: trends in species distribution and antifungal susceptibility. J Clin Microbiol 40: 3551–3557. PubMed PMC

Martin D, Persat F, Piens MA, Picot S (2005) Candida species distribution in bloodstream cultures in Lyon, France, 1998–2001. Eur J Clin Microbiol Infect Dis 24: 329–333. PubMed

Hachem R, Hanna H, Kontoyiannis D, Jiang Y, Raad I (2008) The changing epidemiology of invasive Candidiasis–Candida glabrata and Candida krusei as the leading causes of candidemia in hematologic malignancy. Cancer 112: 2493–2499. 10.1002/cncr.23466 PubMed DOI

Leroy O, Gangneux J, Montravers P, Mira J, Gouin F, et al. (2009) Epidemiology, management, and risk factors for death of invasive Candida infections in critical care: A multicenter, prospective, observational study in France (2005–2006). Crit Care Med: 1612–1618. PubMed

Horn DL, Neofytos D, Anaissie EJ, Fishman JA, Steinbach WJ, et al. (2009) Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry. Clin Infect Dis 48: 1695–1703. 10.1086/599039 PubMed DOI

Pappas P, Rex J, Sobel J, Filler S, Dismukes W, et al. (2004) Guidelines for treatment of candidiasis. Clin Infect Dis: 161–189. PubMed

Ellepola A, Morrison C (2005) Laboratory diagnosis of invasive candidiasis. J Microbiol: 65–84. PubMed

Cuenca-Estrella M, Verweij PE, Arendrup MC, Arikan-Akdagli S, Bille J, et al. (2012) ESCMID* guideline for the diagnosis and management of Candida diseases 2012: diagnostic procedures. Clin Microbiol Infect 18 Suppl 7: 9–18. 10.1111/1469-0691.12038 PubMed DOI

Dendis M, Horvath R, Michalek J, Ruzicka F, Grijalva M, et al. (2003) PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia. Clin Microbiol Infect: 1191–1202. PubMed

Cornet M, Sendid B, Fradin C, Gaillardin C, Poulain D, et al. (2011) Molecular Identification of Closely Related Candida Species Using Two Ribosomal Intergenic Spacer Fingerprinting Methods. J Mol Diagn 13: 12–22. 10.1016/j.jmoldx.2010.11.014 PubMed DOI PMC

Kurtzman CP, Robnett CJ (1997) Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 35: 1216–1223. PubMed PMC

Ciardo DE, Schär G, Böttger EC, Altwegg M, Bosshard PP (2006) Internal transcribed spacer sequencing versus biochemical profiling for identification of medically important yeasts. J Clin Microbiol 44: 77–84. PubMed PMC

Leaw SN, Chang HC, Sun HF, Barton R, Bouchara JP, et al. (2006) Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J Clin Microbiol 44: 693–699. PubMed PMC

Montero C, Shea Y, Jones P, Harrington S, Tooke N, et al. (2008) Evaluation of Pyrosequencing (R) technology for the identification of clinically relevant non-dematiaceous yeasts and related species. Eur J Clinl MicrobiolInfect Dis 27: 821–830. 10.1007/s10096-008-0510-x PubMed DOI PMC

Borman A, Linton C, Oliver D, Palmer M, Szekely A, et al. (2010) Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2. J Clin Microbiol 48: 3648–3653. 10.1128/JCM.01071-10 PubMed DOI PMC

Gharizadeh B, Norberg E, Löffler J, Jalal S, Tollemar J, et al. (2004) Identification of medically important fungi by the Pyrosequencing technology. Mycoses 47: 29–33. PubMed

Loeffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, et al. (2000) Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system. J Clin Microbiol 38: 586–590. PubMed PMC

Pryce TM, Kay ID, Palladino S, Heath CH (2003) Real-time automated polymerase chain reaction (PCR) to detect Candida albicans and Aspergillus fumigatus DNA in whole blood from high-risk patients. Diagn Microbiol Infect Dis 47: 487–496. PubMed

Maaroufi Y, Ahariz N, Husson M, Crokaert F (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. PubMed PMC

Klingspor L, Jalal S (2006) Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR. Clin Microbiol Infect 12: 745–753. PubMed

Fricke S, Fricke C, Schimmelpfennig C, Oelkrug C, Schonfelder U, et al. (2010) A real-time PCR assay for the differentiation of Candida species. J Appl Microbiol 109: 1150–1158. 10.1111/j.1365-2672.2010.04736.x PubMed DOI

Hsu MC, Chen KW, Lo HJ, Chen YC, Liao MH, et al. (2003) Species identification of medically important fungi by use of real-time LightCycler PCR. J Med Microbiol 52: 1071–1076. PubMed

Bergman A, Fernandez V, Holmström KO, Claesson BE, Enroth H (2007) Rapid identification of pathogenic yeast isolates by real-time PCR and two-dimensional melting-point analysis. Eur J Clin Microbiol Infect Dis 26: 813–818. PubMed

Dunyach C, Bertout S, Phelipeau C, Drakulovski P, Reynes J, et al. (2008) Detection and identification of Candida spp. in human serum by LightCycler real-time polymerase chain reaction. Diagn Microbiol Infect Dis 60: 263–271. PubMed

Ahmad S, Khan Z, Mustafa A, Khan Z (2002) Seminested PCR for diagnosis of candidemia: Comparison with culture, antigen detection, and biochemical methods for species identification. J Clin Microbiol 40: 2483–2489. PubMed PMC

Lau A, Sorrell T, Chen S, Stanley K, Iredell J, et al. (2008) Multiplex tandem PCR: a novel platform for rapid detection and identification of fungal pathogens from blood culture specimens. J Clin Microbiol 46: 3021–3027. 10.1128/JCM.00689-08 PubMed DOI PMC

Wang Q, Li J, Wang S, Bai F (2008) Rapid differentiation of phenotypically similar yeast species by single-strand conformation polymorphism analysis of ribosomal DNA. Appl Environl Microb 74: 2604–2611. 10.1128/AEM.02223-07 PubMed DOI PMC

Shepard JR, Addison RM, Alexander BD, Della-Latta P, Gherna M, et al. (2008) Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles. J Clin Microbiol 46: 50–55. PubMed PMC

Marklein G, Josten M, Klanke U, Muller E, Horre R, et al. (2009) Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Fast and Reliable Identification of Clinical Yeast Isolates. J Clin Microbiol 47: 2912–2917. 10.1128/JCM.00389-09 PubMed DOI PMC

Cassagne C, Ranque S, Normand A, Fourquet P, Thiebault S, et al. (2011) Mould Routine Identification in the Clinical Laboratory by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry. Plos One 6 10.1371/journal.pone.0029899 PubMed DOI PMC

Quiles-Melero I, Garcia-Rodriguez J, Gomez-Lopez A, Mingorance J (2012) Evaluation of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry for identification of Candida parapsilosis, C. orthopsilosis and C. metapsilosis. Eur J Clin Microbiol Infect Dis 31: 67–71. 10.1007/s10096-011-1277-z PubMed DOI

Zhao Y, Park S, Kreiswirth BN, Ginocchio CC, Veyret R, et al. (2009) Rapid real-time nucleic Acid sequence-based amplification-molecular beacon platform to detect fungal and bacterial bloodstream infections. J Clin Microbiol 47: 2067–2078. 10.1128/JCM.02230-08 PubMed DOI PMC

Kaleta E, Clark A, Johnson D, Gamage D, Wysocki V, et al. (2011) Use of PCR Coupled with Electrospray Ionization Mass Spectrometry for Rapid Identification of Bacterial and Yeast Bloodstream Pathogens from Blood Culture Bottles. J Clin Microbiol 49: 345–353. 10.1128/JCM.00936-10 PubMed DOI PMC

Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8: 597–608. PubMed

Cheng JC, Huang CL, Lin CC, Chen CC, Chang YC, et al. (2006) Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clin Chem 52: 1997–2004. PubMed

Stephens AJ, Inman-Bamber J, Giffard PM, Huygens F (2008) High-resolution melting analysis of the spa repeat region of Staphylococcus aureus. Clin Chem 54: 432–436. 10.1373/clinchem.2007.093658 PubMed DOI

Won H, Rothman R, Ramachandran P, Hsieh YH, Kecojevic A, et al. (2010) Rapid identification of bacterial pathogens in positive blood culture bottles by use of a broad-based PCR assay coupled with high-resolution melt analysis. J Clin Microbiol 48: 3410–3413. 10.1128/JCM.00718-10 PubMed DOI PMC

Slany M, Vanerkova M, Nemcova E, Zaloudikova B, Ruzicka F, et al. (2010) Differentiation of Staphylococcus spp. by high-resolution melting analysis. Can J Microbiol 56: 1040–1049. 10.1139/W10-091 PubMed DOI

Steer PA, Kirkpatrick NC, O'Rourke D, Noormohammadi AH (2009) Classification of fowl adenovirus serotypes by use of high-resolution melting-curve analysis of the hexon gene region. J Clin Microbiol 47: 311–321. 10.1128/JCM.01567-08 PubMed DOI PMC

Tajiri-Utagawa E, Hara M, Takahashi K, Watanabe M, Wakita T (2009) Development of a rapid high-throughput method for high-resolution melting analysis for routine detection and genotyping of noroviruses. J Clin Microbiol 47: 435–440. 10.1128/JCM.01247-08 PubMed DOI PMC

Plachy R, Hamal P, Raclavsky V (2005) McRAPD as a new approach to rapid and accurate identification of pathogenic yeasts. J Microbiol Meth 60: 107–113. PubMed

Trtkova J, Pavlicek P, Ruskova L, Hamal P, Koukalova D, et al. (2009) Performance of optimized McRAPD in identification of 9 yeast species frequently isolated from patient samples: potential for automation. BMC Microbiol 9 10.1186/1471-2180-9-281 PubMed DOI PMC

Hrncirova K, Lengerova M, Kocmanova I, Racil Z, Volfova P, et al. (2010) Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis. J Clin Microbiol: 3392–3394. PubMed PMC

Mandviwala T, Shinde R, Kalra A, Sobel J, Akins R (2010) High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons. J Mol Diagn: 91–101. PubMed PMC

Arancia S, Sandini S, De Bernardis F, Fortini D (2011) Rapid, simple, and low-cost identification of Candida species using high-resolution melting analysis. Diagn Microbiol Infect Dis 69: 283–285. 10.1016/j.diagmicrobio.2010.10.003 PubMed DOI

Somogyvari F, Horvath A, Serly J, Majoros H, Vagvolgyi C, et al. (2012) Detection of Invasive Fungal Pathogens by Real-time PCR and High-resolution Melting Analysis. In Vivo 26: 979–983. PubMed

Goldschmidt P, Degorge S, Sarria P, Benallaoua D, Semoun O, et al. (2012) New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings. Plos One 7 10.1371/journal.pone.0051204 PubMed DOI PMC

Decat E, Van Mechelen E, Saerens B, Vermeulen SJ, Boekhout T, et al. (2013) Rapid and accurate identification of isolates of Candida species by melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA). Res Microbiol 164: 110–117. 10.1016/j.resmic.2012.10.017 PubMed DOI

Alnuaimi AD, Wiesenfeld D, O'Brien-Simpson NM, Reynolds EC, Peng B, et al. (2014) The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis. Mol Oral Microbiol 29: 117–130. 10.1111/omi.12050 PubMed DOI

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410. PubMed

CBS-KNAW Fungal Biodiversity Centre CBS Filamentous Fungi Database. Utrecht, The Netherlands.

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599. PubMed

Dwight Z, Palais R, Wittwer CT (2011) uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics 27: 1019–1020. 10.1093/bioinformatics/btr065 PubMed DOI

Lockhart SR, Messer SA, Pfaller MA, Diekema DJ (2008) Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis. J Clin Microbiol 46: 2659–2664. 10.1128/JCM.00803-08 PubMed DOI PMC

Cantón E, Pemán J, Quindós G, Eraso E, Miranda-Zapico I, et al. (2011) Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia. Antimicrob Agents Chemother 55: 5590–5596. 10.1128/AAC.00466-11 PubMed DOI PMC

Desnos-Ollivier M, Ragon M, Robert V, Raoux D, Gantier J, et al. (2008) Debaryomyces hansenii (Candida famata), a rare human fungal pathogen often misidentified as Pichia guilliermondii (Candida guilliermondii). J Clin Microbiol 46: 3237–3242. 10.1128/JCM.01451-08 PubMed DOI PMC

Hamal P, Ostransky J, Dendis M, Horvath R, Ruzicka F, et al. (2008) A case of endocarditis caused by the yeast Pichia fabianii with biofilm production and developed in vitro resistance to azoles in the course of antifungal treatment. Med Mycol 46: 601–605. 10.1080/13693780802078180 PubMed DOI

Valenza G, Valenza R, Brederlau J, Frosch M, Kurzai O (2006) Identification of Candida fabianii as a cause of lethal septicaemia. Mycoses 49: 331–334. PubMed

Staib P, Morschhäuser J (1999) Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis. Mycoses 42: 521–524. PubMed

Pincus DH, Coleman DC, Pruitt WR, Padhye AA, Salkin IF, et al. (1999) Rapid identification of Candida dubliniensis with commercial yeast identification systems. J Clin Microbiol 37: 3533–3539. PubMed PMC

Ellepola AN, Hurst SF, Elie CM, Morrison CJ (2003) Rapid and unequivocal differentiation of Candida dubliniensis from other Candida species using species-specific DNA probes: comparison with phenotypic identification methods. Oral Microbiol Immunol 18: 379–388. PubMed

Chryssanthou E, Fernandez V, Petrini B (2007) Performance of commercial latex agglutination tests for the differentiation of Candida dubliniensis and Candida albicans in routine diagnostics. APMIS 115: 1281–1284. PubMed

Capability of Fluorescent Capillary Electrophoresis To Distinguish Species of the Candida parapsilosis Complex

The Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery

Fluorescent Capillary Electrophoresis Is Superior to Culture in Detecting Candida Species from Samples of Urinary Catheters and Ureteral Stents with Mono- or Polyfungal Biofilm Growth

Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections