Early diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex.

Centre for Cardiovascular Surgery and Transplantation Brno Genetics Laboratory Brno Czech Republic

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

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

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

Zobrazit více v PubMed

Ito JI, Kriengkauykiat J, Dadwal SS, Arfons LM, Lazarus HM. 2010. Approaches to the early treatment of invasive fungal infection. Leuk Lymphoma 51:1623–1631. doi:10.3109/10428194.2010.496504. PubMed DOI

Chandrasekar P. 2010. Diagnostic challenges and recent advances in the early management of invasive fungal infections. Eur J Haematol 84:281–290. doi:10.1111/j.1600-0609.2009.01391.x. PubMed DOI

Hobson RP. 2003. The global epidemiology of invasive Candida infections—is the tide turning? J Hosp Infect 55:159–168. doi:10.1016/j.jhin.2003.08.012. PubMed DOI

Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. 2004. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 39:309–317. doi:10.1086/421946. PubMed DOI

Nawrot U, Kowalska-Krochmal B, Sulik-Tyszka B, Kozak M, Świętek K, Pajączkowska M, Piątkowska E, Rosiak D, Swoboda-Kopeć E. 2015. Evaluation of blood culture media for the detection of fungi. Eur J Clin Microbiol Infect Dis 34:161–167. doi:10.1007/s10096-014-2218-4. PubMed DOI PMC

Kollef M, Micek S, Hampton N, Doherty JA, Kumar A. 2012. Septic shock attributed to Candida infection: importance of empiric therapy and source control. Clin Infect Dis 54:1739–1746. doi:10.1093/cid/cis305. PubMed DOI

Bassetti M, Righi E, Ansaldi F, Merelli M, Cecilia T, De Pascale G, Diaz-Martin A, Luzzati R, Rosin C, Lagunes L, Trecarichi EM, Sanguinetti M, Posteraro B, Garnacho-Montero J, Sartor A, Rello J, Rocca GD, Antonelli M, Tumbarello M. 2014. A multicenter study of septic shock due to candidemia: outcomes and predictors of mortality. Intensive Care Med 40:839–845. doi:10.1007/s00134-014-3310-z. PubMed DOI

Nucci M, Marr KA. 2005. Emerging fungal diseases. Clin Infect Dis 41:521–526. doi:10.1086/432060. PubMed DOI

Lass-Flörl C. 2009. The changing face of epidemiology of invasive fungal disease in Europe. Mycoses 52:197–205. doi:10.1111/j.1439-0507.2009.01691.x. PubMed DOI

Avni T, Leibovici L, Paul M. 2011. PCR diagnosis of invasive candidiasis: systematic review and meta-analysis. J Clin Microbiol 49:665–670. doi:10.1128/JCM.01602-10. PubMed DOI PMC

Bader O. 2013. MALDI-TOF-MS-based species identification and typing approaches in medical mycology. Proteomics 13:788–799. doi:10.1002/pmic.201200468. PubMed DOI

Majoros L, Kardos G, Belak A, Maraz A, Asztalos L, Csanky E, Barta Z, Szabo B. 2003. Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures. J Clin Microbiol 41:5250–5253. doi:10.1128/JCM.41.11.5250-5253.2003. PubMed DOI PMC

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

Pulcrano G, Iula VD, Vollaro A, Tucci A, Cerullo M, Esposito M, Rossano F, Catania MR. 2013. Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections. J Microbiol Methods 94:262–266. doi:10.1016/j.mimet.2013.07.001. PubMed DOI

Cuenca-Estrella M, Verweij PE, Arendrup MC, Arikan-Akdagli S, Bille J, Donnelly JP, Jensen HE, Lass-Flörl C, Richardson MD, Akova M, Bassetti M, Calandra T, Castagnola E, Cornely OA, Garbino J, Groll AH, Herbrecht R, Hope WW, Kullberg BJ, Lortholary O, Meersseman W, Petrikkos G, Roilides E, Viscoli C, Ullmann AJ. 2012. ECCMID guideline for the diagnosis and management of Candida diseases 2012: diagnostic procedures. Clin Microbiol Infect 18:9–18. doi:10.1111/1469-0691.12038. PubMed DOI

Gavaldà J, Meije Y, Fortún J, Roilides E, Saliba F, Lortholary O, Muñoz P, Grossi P, Cuenca-Estrella M. 2014. Invasive fungal infections in solid organ transplant recipients. Clin Microbiol Infect 20(Suppl 7):27–48. doi:10.1111/1469-0691.12660. PubMed DOI

Lau A, Halliday C, Chen SCA, Playford EG, Stanley K, Sorrell TC. 2010. Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR. J Clin Microbiol 48:811–816. doi:10.1128/JCM.01650-09. PubMed DOI PMC

Nguyen MH, Wissel MC, Shields RK, Salomoni MA, Hao B, Press EG, Shields RM, Cheng S, Mitsani D, Vadnerkar A, Silveira FP, Kleiboeker SB, Clancy CJ. 2012. Performance of Candida real-time polymerase chain reaction, β-glucan assay, and blood cultures in the diagnosis of invasive candidiasis. Clin Infect Dis 54:1240–1248. doi:10.1093/cid/cis200. PubMed DOI

Alanio A, Bretagne S. 2014. Difficulties with molecular diagnostic tests for mould and yeast infections: where do we stand? Clin Microbiol Infect 20:36–41. doi:10.1111/1469-0691.12617. PubMed DOI

Guiver M, Levi K, Oppenheim BA. 2001. Rapid identification of Candida species by TaqMan PCR. J Clin Pathol 54:362–366. doi:10.1136/jcp.54.5.362. PubMed DOI PMC

Dunyach C, Bertout S, Phelipeau C, Drakulovski P, Reynes J, Mallié M. 2008. Detection and identification of Candida spp. in human serum by LightCycler real-time polymerase chain reaction. Diagn Microbial Infect Dis 60:263–271. doi:10.1016/j.diagmicrobio.2007.09.014. PubMed DOI

Fricke S, Fricke C, Schimmelpfennig C, Oelkrug C, Schönfelder U, Blatz R, Zilch C, Faber S, Hilger N, Ruhnke M, Rodloff AC. 2010. A real-time PCR assay for the differentiation of Candida species. J Appl Microbiol 109:1150–1158. doi:10.1111/j.1365-2672.2010.04736.x. PubMed DOI

Leaw SN, Chang HC, Sun HF, Barton R, Bouchara JP, Chang TC. 2006. Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J Clin Microbiol 44:693–699. doi:10.1128/JCM.44.3.693-699.2006. PubMed DOI PMC

Williams DW, Wilson MJ, Lewis MAO, Potts AJC. 1995. Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer region of ribosomal DNA. J Clin Microbiol 33:2476–2479. 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. doi:10.1016/j.diagmicrobio.2010.10.003. PubMed DOI

Alnuaimi AD, Wiesenfeld D, O'Brien-Simpson NM, Reynolds EC, Peng B, McCullough MJ. 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. doi:10.1111/omi.12050. PubMed DOI

Nemcova E, Cernochova M, Ruzicka F, Malisova B, Freiberger T, Nemec P. 2015. Rapid identification of medically important Candida isolates using high resolution melting analysis. PLoS One 10:e0116940. doi:10.1371/journal.pone.0116940. PubMed DOI PMC

Spiess B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, Morz H, Hehlmann R, Buchheidt D. 2007. DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients. J Clin Microbiol 45:3743–3753. doi:10.1128/JCM.00942-07. PubMed DOI PMC

Campa D, Tavanti A, Gemignani F, Mogavero CS, Bellini I, Bottari F, Barale R, Landi S, Senesi S. 2008. DNA microarray based on arrayed-primer extension technique for identification of pathogenic fungi responsible for invasive and superficial mycoses. J Clin Microbiol 46:909–915. doi:10.1128/JCM.01406-07. PubMed DOI PMC

Gharizadeh B, Norberg E, Loffler J, Jalal S, Tollemar J, Einsele H, Klingspor L, Nyren P. 2004. Identification of medically important fungi by the pyrosequencing technology. Mycoses 47:29–33. doi:10.1046/j.1439-0507.2003.00949.x. PubMed DOI

Turenne ChY, Sanche SE, Hoban DJ, Karlowsky JA, Kabani AM. 1999. Rapid identification of fungi by using ITS2 genetic region and an automated fluorescent capillary electrophoresis system. J Clin Microbiol 37:1846–1851. PubMed PMC

De Baere T, Claeys G, Swinne D, Massonet C, Verschraegen G, Muylaert A, Vaneechoutte M. 2002. Identification of cultured isolates of clinically important species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region. BMC Microbiol 2:21. doi:10.1186/1471-2180-2-21. PubMed DOI PMC

Landlinger C, Bašková L, Preuner S, Willinger B, Buchta V, Lion T. 2009. Identification of fungal species by fragment length analysis of the internally transcribed spacer 2 region. Eur J Clin Microbiol Infect Dis 28:613–622. doi:10.1007/s10096-008-0683-3. PubMed DOI

Liu CY, Zhang J, Xu X, Chen J. 2011. Prediction of DNA separation by capillary electrophoresis with polymer additives. J Chromatogr Sci 49:310–315. doi:10.1093/chrsci/49.4.310. PubMed DOI

Dendis M, Horváth R, Michálek J, Růžička F, Grijalva M, Bartoš M, Benedík J. 2003. PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia. Clin Microbiol Infect 9:1191–1202. doi:10.1111/j.1469-0691.2003.00719.x. PubMed DOI

Chen YC, Eisner JD, Kattar MM, Rassoullian-Barrett SL, Lafe K, Yarfitz SL, Limaye AP, Cookson BT. 2000. Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes. J Clin Microbiol 38:2302–2310. PubMed PMC

Smith JR, Carpten JD, Brownstein MJ, Ghosh S, Magnuson VL, Gilbert DA, Trent JM, Collins FS. 1995. Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase. Genome Res 5:312–317. doi:10.1101/gr.5.3.312. PubMed DOI

Vaughan-Martini A, Kurtzman CP, Meyer SA, O'Neill EB. 2005. Two new species in the Pichia guilliermondii clade: Pichia carribica sp. nov., the ascosporic state of Candida fermentati and Candida carpophila comb. nov. FEMS Yeast Res 5:463–469. doi:10.1016/j.femsyr.2004.10.008. PubMed DOI

Romeo O, Criseo G. 2011. Candida africana and its closest relatives. Mycoses 54:475–486. doi:10.1111/j.1439-0507.2010.01939.x. PubMed DOI

Alonso-Vargas R, Elorduy L, Eraso E, Francisco Cano J, Guarro J, Pontón J, Quindós G. 2008. Isolation of Candida africana, probable atypical strains of Candida albicans, from a patient with vaginitis. Med Mycol 46:167–170. doi:10.1080/13693780701633101. PubMed DOI

Horn DL, Neofytos D, Anaissie EJ, Fishman JA, Steinbach WJ, Olyaei AJ, Marr KA, Pfaller MA, Chang CH, Webster KM. 2009. Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry. Clin Infect Dis 48:1695–1703. doi:10.1086/599039. PubMed DOI

Pappas PG, Kauffman CA, Andes D, Benjamin DK, Calandra TF, Edwards JE, Filler SG, Fisher JF, Kullberg BJ, Ostrosky-Zeichner L, Reboli AC, Rex JH, Walsh TJ, Sobel JD. 2009. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 48:503–535. doi:10.1086/596757. PubMed DOI PMC

Cantón E, Peman J, Quindos G, Eraso E, Miranda-Zapico I, Alvarez M, Merino P, Campos-Herrero I, Marco F, de la Pedrosa EGG, Yague G, Guna R, Rubio C, Miranda C, Pazos C, Velasco D. 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. doi:10.1128/AAC.00466-11. PubMed DOI PMC

Marklein G, Josten M, Klanke U, Müller E, Horré R, Maier T, Wenzel T, Kostrzewa M, Bierbaum G, Hoerauf A, Sahl H-G. 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. doi:10.1128/JCM.00389-09. PubMed DOI PMC

Rickerts V, McCormick Smith I, Mousset S, Kommedal O, Fredricks DN. 2013. Deciphering the aetiology of a mixed fungal infection by broad-range PCR with sequencing and fluorescence in situ hybridisation. Mycoses 56:681–686. doi:10.1111/myc.12083. PubMed DOI

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

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