Rapid and accurate identification of species belonging to the complex by real-time PCR and melting curve analysis Free

Abstract

is the second most frequent species isolated from blood cultures. Since 2005, has been divided into three distinct species based on genetic traits: , and . The aim of this study was to develop a rapid real-time PCR assay able to distinguish these closely related species via a melting curve analysis. This identification method was optimized by using reference strains and well-characterized clinical isolates of species. A single set of consensus primers was designed to amplify a 184 bp portion of the gene in order to identify species based on the unique melt profile resulting from DNA sequence variations from each species of the complex. PCR products were detected with SYBR Green fluorescent dye and identification was established by melting curve analysis. For validation of the technique, a total of 116 clinical isolates, phenotypically identified as , were tested by real-time PCR and results were further compared with PCR-RFLP patterns of the gene, used as the reference method. The melting curve analysis of amplified DNA could differentiate between (83.5 °C), (82.9 °C) and (82.1 °C), with a sensitivity and specificity comparable to those of the reference method. One hundred and fourteen and two isolates were identified among the clinical isolates. This method provides a simple, rapid and reliable identification of species belonging to the complex. This novel approach could be helpful for clinical and epidemiological investigations.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.026633-0
2011-04-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/60/4/477.html?itemId=/content/journal/jmm/10.1099/jmm.0.026633-0&mimeType=html&fmt=ahah

References

  1. Almirante B., Rodríguez D., Cuenca-Estrella M., Almela M., Sanchez F., Ayats J., Alonso-Tarres C., Rodriguez-Tudela J. L., Pahissa A. the Barcelona Candidemia Project Study Group 2006; Epidemiology, risk factors, and prognosis of Candida parapsilosis bloodstream infections: case-control population-based surveillance study of patients in Barcelona, Spain, from 2002 to 2003. J Clin Microbiol 44:1681–1685 [CrossRef]
    [Google Scholar]
  2. Asadzadeh M., Ahmad S., Al-Sweih N., Khan Z. U. 2009; Rapid molecular differentiation and genotypic heterogeneity among Candida parapsilosis and Candida orthopsilosis strains isolated from clinical specimens in Kuwait. J Med Microbiol 58:745–752 [CrossRef]
    [Google Scholar]
  3. Bassetti M., Ansaldi F., Nicolini L., Malfatto E., Molinari M. P., Mussap M., Rebesco B., Bobbio Pallavicini F., Icardi G., Viscoli C. 2009; Incidence of candidaemia and relationship with fluconazole use in an intensive care unit. J Antimicrob Chemother 64:625–629 [CrossRef]
    [Google Scholar]
  4. Borman A. M., Linton C. J., Oliver D., Palmer M. D., Szekely A., Odds F. C., Johnson E. M. 2009; Pyrosequencing analysis of 20 nucleotides of internal transcribed spacer 2 discriminates Candida parapsilosis , Candida metapsilosis , and Candida orthopsilosis . J Clin Microbiol 47:2307–2310 [CrossRef]
    [Google Scholar]
  5. Filioti J., Spiroglou K., Panteliadis C. P., Roilides E. 2007; Invasive candidiasis in pediatric intensive care patients: epidemiology, risk factors, management, and outcome. Intensive Care Med 33:1272–1283 [CrossRef]
    [Google Scholar]
  6. Forrest G. N., Weekes E., Johnson J. K. 2008; Increasing incidence of Candida parapsilosis candidemia with caspofungin usage. J Infect 56:126–129 [CrossRef]
    [Google Scholar]
  7. Gomez-Lopez A., Alastruey-Izquierdo A., Rodriguez D., Almirante B., Pahissa A., Rodriguez-Tudela J. L., Cuenca-Estrella M. the Barcelona Candidemia Project Study Group 2008; Prevalence and susceptibility profile of Candida metapsilosis and Candida orthopsilosis : results from population-based surveillance of candidemia in Spain. Antimicrob Agents Chemother 52:1506–1509 [CrossRef]
    [Google Scholar]
  8. Hensgens L. A., Tavanti A., Mogavero S., Ghelardi E., Senesi S. 2009; AFLP genotyping of Candida metapsilosis clinical isolates: evidence for recombination. Fungal Genet Biol 46:750–758 [CrossRef]
    [Google Scholar]
  9. Khan Z., Mustafa A. S., Alam F. F. 2009; Real-time LightCycler polymerase chain reaction and melting temperature analysis for identification of clinically important Candida spp. J Microbiol Immunol Infect 42:290–295
    [Google Scholar]
  10. Kuhn D. M., Chandra J., Mukherjee P. K., Ghannoum M. A. 2002; Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect Immun 70:878–888 [CrossRef]
    [Google Scholar]
  11. Lockhart S. R., Messer S. A., Pfaller M. A., Diekema D. J. 2008a; Lodderomyces elongisporus masquerading as Candida parapsilosis as a cause of bloodstream infections. J Clin Microbiol 46:374–376 [CrossRef]
    [Google Scholar]
  12. Lockhart S. R., Messer S. A., Pfaller M. A., Diekema D. J. 2008b; 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 [CrossRef]
    [Google Scholar]
  13. Playford E. G., Nimmo G. R., Tilse M., Sorrell T. C. 2010; Increasing incidence of candidaemia: long-term epidemiological trends. Queensland, Australia: 1999–2008 J Hosp Infect 76:46–51 [CrossRef]
    [Google Scholar]
  14. Růžička F., Holá V., Votava M., Tejkalová R. 2007; Importance of biofilm in Candida parapsilosis and evaluation of its susceptibility to antifungal agents by colorimetric method. Folia Microbiol (Praha 52:209–214 [CrossRef]
    [Google Scholar]
  15. Tavanti A., Davidson A. D., Gow N. A., Maiden M. C., Odds F. C. 2005; Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 43:284–292 [CrossRef]
    [Google Scholar]
  16. Tavanti A., Hensgens L. A., Ghelardi E., Campa M., Senesi S. 2007; Genotyping of Candida orthopsilosis clinical isolates by amplification fragment length polymorphism reveals genetic diversity among independent isolates and strain maintenance within patients. J Clin Microbiol 45:1455–1462 [CrossRef]
    [Google Scholar]
  17. Tavanti A., Hensgens L. A., Mogavero S., Majoros L., Senesi S., Campa M. 2010; Genotypic and phenotypic properties of Candida parapsilosis sensu strictu strains isolated from different geographic regions and body sites. BMC Microbiol 10:203 [CrossRef]
    [Google Scholar]
  18. Trofa D., Gácser A., Nosanchuk J. D. 2008; Candida parapsilosis , an emerging fungal pathogen. Clin Microbiol Rev 21:606–625 [CrossRef]
    [Google Scholar]
  19. van Asbeck E. C., Huang Y.-C., Markham A. N., Clemons K. V., Stevens D. A. 2007; Candida parapsilosis fungemia in neonates: genotyping results suggest healthcare workers hands as source, and review of published studies. Mycopathologia 164:287–293 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.026633-0
Loading
/content/journal/jmm/10.1099/jmm.0.026633-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed