, Volume 177, Issue 5–6, pp 251–261 | Cite as

Multi-probe Real-Time PCR Identification of Four Common Candida Species in Blood Culture Broth

  • Suporn Foongladda
  • Nanthanida Mongkol
  • Pornphan Petlum
  • Methee Chayakulkeeree


We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.


Candida Real-time PCR Probes Blood Identification Candidemia 



This work was supported by the Siriraj Research Development Fund, Mahidol University. S. Foongladda and M. Chayakulkeeree were supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University.


  1. 1.
    Mullen CA, El-Baki HA, Samir H, Tarrand JJ, Rolston KV. Non-albicans Candida is the most common cause of candidemia in pediatric cancer patients. Support Care Cancer. 2003;11:321–5.PubMedGoogle Scholar
  2. 2.
    Fidel PL Jr, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev. 1999;12:80–96.PubMedCentralPubMedGoogle Scholar
  3. 3.
    Krcmery V, Barnes AJ. Non-albicans Candida spp. causing fungaemia: pathogenicity and antifungal resistance. J Hosp Infect. 2002;50:243–60.PubMedCrossRefGoogle Scholar
  4. 4.
    Trofa D, Gacser A, Nosanchuk JD. Candida parapsilosis, an emerging fungal pathogen. Clin Microbiol Rev. 2008;21:606–25.PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Pfaller MA, Diekema DJ. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol. 2010;2012(50):2846–56.Google Scholar
  6. 6.
    Freydiere AM, Odds FC. Commercial kits for yeast identification: concerns for standardisation. Eur J Clin Microbiol Infect Dis. 2001;20:366–7.PubMedGoogle Scholar
  7. 7.
    Freydiere AM, Guinet R, Boiron P. Yeast identification in the clinical microbiology laboratory: phenotypical methods. Med Mycol. 2001;39:9–33.PubMedCrossRefGoogle Scholar
  8. 8.
    Ellepola AN, Morrison CJ. Laboratory diagnosis of invasive candidiasis. J Microbiol. 2005;43:65–84.PubMedGoogle Scholar
  9. 9.
    Elie CM, Lott TJ, Reiss E, Morrison CJ. Rapid identification of Candida species with species-specific DNA probes. J Clin Microbiol. 1998;36:3260–5.PubMedCentralPubMedGoogle Scholar
  10. 10.
    Bougnoux M, Dupont C, Mateo J, Saulnier P, Faivre V, Payen D, Nicolas-Chanoine M. Serum is more suitable than whole blood for diagnosis of systemic candidiasis by nested PCR. J Clin Microbiol. 1999;37:925–30.PubMedCentralPubMedGoogle Scholar
  11. 11.
    Guiver M, Levi K, Oppenheim BA. Rapid identification of Candida species by TaqMan PCR. J Clin Pathol. 2001;54:362–6.PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Maaroufi Y, Heymans C, De Bruyne JM, Duchateau V, Rodriguez-Villalobos H, Aoun M, Crokaert F. Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J Clin Microbiol. 2003;41:3293–8.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Schabereiter-Gurtner C, Selitsch B, Rotter ML, Hirschl AM, Willinger B. Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens. J Clin Microbiol. 2007;45:906–14.PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Fricke S, Fricke C, Schimmelpfennig C, Oelkrug C, Schönfelder U, Blatz R, Zilch C, Faber S, Hilger N, Ruhnke M, Rodloff AC. A real-time PCR assay for the differentiation of Candida species. J Appl Microbiol. 2010;109:1150–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Selvarangan R, Bui U, Limaye AP, Cookson BT. Rapid identification of commonly encountered Candida species directly from blood culture bottles. J Clin Microbiol. 2003;41:5660–4.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Gherna M, Merz WG. Identification of Candida albicans and Candida glabrata within 1.5 hours directly from positive blood culture bottles with a shortened peptide nucleic acid fluorescence in situ hybridization protocol. J Clin Microbiol. 2009;47:247–8.PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Lagacé-Wiens PR, Adam HJ, Karlowsky JA, Nichol KA, Pang PF, Guenther J, Webb AA, Miller C, Alfa MJ. Identification of blood culture isolates directly from positive blood cultures by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry and a commercial extraction system: analysis of performance, cost, and turnaround time. J Clin Microbiol. 2012;50:3324–8.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Foongladda S, Pholwat S, Eampokalap B, Kiratisin P, Sutthent R. Multi-probe real-time PCR identification of common mycobacterium species in blood culture broth. J Mol Diagn. 2009;11:42–8.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Quiles-Melero I, García-Rodriguez J, Romero-Gómez MP, Gómez-Sánchez P, Mingorance J. Rapid identification of yeasts from positive blood culture bottles by pyrosequencing. Eur J Clin Microbiol Infect Dis. 2011;30:21–4.PubMedCrossRefGoogle Scholar
  20. 20.
    Fenn JP, Billetdeaux E, Segal H, Skodack-Jones L, Padilla PE, Bale M, Carroll K. Comparison of four methodologies for rapid and cost-effective identification of Candida glabrata. J Clin Microbiol. 1999;37:3387–9.PubMedCentralPubMedGoogle Scholar
  21. 21.
    Vallini G, Frassinetti S, Scorzetti G. Candida aquaetextoris sp. nov., a new species of yeast occurring in sludge from a textile industry wastewater treatment plant in Tuscany, Italy. Int J Syst Bacteriol. 1997;47:336–40.PubMedCrossRefGoogle Scholar
  22. 22.
    Suh SO, Blackwell M. Three new beetle-associated yeast species in the Pichia guilliermondii clade. FEMS Yeast Res. 2004;5:87–95.PubMedCrossRefGoogle Scholar
  23. 23.
    Suh SO, McHugh JV, Pollock DD, Blackwell M. The beetle gut: a hyperdiverse source of novel yeasts. Mycol Res. 2005;109:261–5.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Suporn Foongladda
    • 1
  • Nanthanida Mongkol
    • 1
  • Pornphan Petlum
    • 1
  • Methee Chayakulkeeree
    • 2
  1. 1.Department of Microbiology, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand
  2. 2.Department of Medicine, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand

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