1887

Abstract

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive species. Three TaqMan-based real-time PCR assays were developed to identify up to six species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify , , , , and ; these are the most commonly encountered species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 18S rRNA sequences conserved in the four species. The FLC-resistant species and were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (=33 sets) and the results were 100 % concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of and in comparison with a conventional phenotypic method, from ∼72 to <3 h, and consequently allows optimization of the antifungal regimen at an earlier stage.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.47149-0
2007-07-01
2019-10-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/56/7/964.html?itemId=/content/journal/jmm/10.1099/jmm.0.47149-0&mimeType=html&fmt=ahah

References

  1. Alexander, B. D., Ashley, E. D., Reller, L. B. & Reed, S. D. ( 2006; ). Cost savings with implementation of PNA FISH testing for identification of Candida albicans in blood cultures. Diagn Microbiol Infect Dis 54, 277–282.[CrossRef]
    [Google Scholar]
  2. Aliyu, S. H., Enoch, D. A., Abubakar, I. I., Ali, R., Carmichael, A. J., Farrington, M. & Lever, M. L. ( 2006; ). Candidaemia in a large teaching hospital: a clinical audit. QJM 99, 655–663.[CrossRef]
    [Google Scholar]
  3. Beck-Sague, C. & Jarvis, W. R. ( 1993; ). Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990. J Infect Dis 167, 1247–1251.[CrossRef]
    [Google Scholar]
  4. Chang, H. C., Leaw, S. N., Huang, A. H., Wu, T. L. & Chang, T. C. ( 2001; ). Rapid identification of yeasts in positive blood cultures by a multiplex PCR method. J Clin Microbiol 39, 3466–3471.[CrossRef]
    [Google Scholar]
  5. Das, S., Brown, T. M., Kellar, K. L., Holloway, B. P. & Morrison, C. J. ( 2006; ). DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system. FEMS Immunol Med Microbiol 46, 244–250.[CrossRef]
    [Google Scholar]
  6. Dooley, D. P., Beckius, M. L. & Jeffrey, B. S. ( 1994; ). Misidentification of clinical yeast isolates by using the updated Vitek Yeast Biochemical Card. J Clin Microbiol 32, 2889–2892.
    [Google Scholar]
  7. Einsele, H., Hebart, H., Roller, G., Loffler, J., Rothenhofer, I., Muller, C. A., Bowden, R. A., van Burik, J., Engelhard, D. & other authors ( 1997; ). Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 35, 1353–1360.
    [Google Scholar]
  8. Elie, C. M., Lott, T. J., Reiss, E. & Morrison, C. J. ( 1998; ). Rapid identification of Candida species with species-specific DNA probes. J Clin Microbiol 36, 3260–3265.
    [Google Scholar]
  9. Ellepola, A. N. & Morrison, C. J. ( 2005; ). Laboratory diagnosis of invasive candidiasis. J Microbiol 43, 65–84.
    [Google Scholar]
  10. Espinel-Ingroff, A., Stockman, L., Roberts, G., Pincus, D., Pollack, J. & Marler, J. ( 1998; ). Comparison of RapID yeast plus system with API 20C system for identification of common, new, and emerging yeast pathogens. J Clin Microbiol 36, 883–886.
    [Google Scholar]
  11. Fadda, G. ( 2000; ). Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from Candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples. J Clin Microbiol 38, 1609–1614.
    [Google Scholar]
  12. Forrest, G. N., Mankes, K., Jabra-Rizk, M. A., Weekes, E., Johnson, J. K., Lincalis, D. P. & Venezia, R. A. ( 2006; ). Peptide nucleic acid fluorescence in situ hybridization-based identification of Candida albicans and its impact on mortality and antifungal therapy costs. J Clin Microbiol 44, 3381–3383.[CrossRef]
    [Google Scholar]
  13. Fredricks, D. N. & Relman, D. A. ( 1998; ). Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J Clin Microbiol 36, 2810–2816.
    [Google Scholar]
  14. Fridkin, S. K. & Jarvis, W. R. ( 1996; ). Epidemiology of nosocomial fungal infections. Clin Microbiol Rev 9, 499–511.
    [Google Scholar]
  15. Fujita, S., 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.
    [Google Scholar]
  16. Garey, K. W., Rege, M., Pai, M. P., Mingo, D. E., Suda, K. J., Turpin, R. S. & Bearden, D. T. ( 2006; ). Time to initiation of fluconazole therapy impacts mortality in patients with candidemia: a multi-institutional study. Clin Infect Dis 43, 25–31.[CrossRef]
    [Google Scholar]
  17. 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.[CrossRef]
    [Google Scholar]
  18. Luo, G. & Mitchell, T. G. ( 2002; ). Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR. J Clin Microbiol 40, 2860–2865.[CrossRef]
    [Google Scholar]
  19. Maaroufi, Y., Heymans, C., De-Bruyne, J. M., Duchateau, V., Rodriguez-Villalobos, H., Aoun, M. & Crokaert, F. ( 2003; ). Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J Clin Microbiol 41, 3293–3298.[CrossRef]
    [Google Scholar]
  20. McMullan, R., McClurg, R., Xu, J., Moore, J. E., Millar, B. C., Crowe, M. & Hedderwick, S. ( 2002; ). Trends in the epidemiology of Candida bloodstream infections in Northern Ireland between January 1984 and December 2000. J Infect 45, 25–28.[CrossRef]
    [Google Scholar]
  21. 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.
    [Google Scholar]
  22. Moreira-Oliveira, M. S., Mikami, Y., Miyaji, M., Imai, T., Schreiber, A. Z. & Moretti, M. L. ( 2005; ). Diagnosis of candidemia by polymerase chain reaction and blood culture: prospective study in a high-risk population and identification of variables associated with development of candidemia. Eur J Clin Microbiol Infect Dis 24, 721–726.[CrossRef]
    [Google Scholar]
  23. Oliveira, K., Haase, G., Kurtzman, C., Hyldig-Nielsen, J. J. & Stender, H. ( 2001; ). Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes. J Clin Microbiol 39, 4138–4141.[CrossRef]
    [Google Scholar]
  24. Park, S., Wong, M., Marras, S. A. E., Cross, E. W., Kiehn, T. E., Chaturvedi, V., Tyagi, S. & Perlin, D. S. ( 2000; ). Rapid identification of Candida dubliniensis using a species-specific molecular beacon. J Clin Microbiol 38, 2829–2836.
    [Google Scholar]
  25. Pfaller, M. A. ( 1996; ). Nosocomial candidiasis: emerging species, reservoirs, and modes of transmission. Clin Infect Dis 22 (Suppl. 2), S89–S94.[CrossRef]
    [Google Scholar]
  26. Pfaller, M. A., Messer, S. A., Hollis, R. J., Jones, R. N., Doern, G. V., Brandt, M. E. & Hajjeh, R. A. ( 1999; ). Trends in species distribution and susceptibility to fluconazole among blood stream isolates of Candida species in the United States. Diagn Microbiol Infect Dis 33, 217–222.[CrossRef]
    [Google Scholar]
  27. Pfaller, M. A., Diekema, D. J., Jones, R. N., Sader, H. S., Fluit, A. C., Hollis, R. J. & Messer, S. A. ( 2001; ). International surveillance of bloodstream infections due to Candida species: frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole, and voriconazole of isolates collected from 1997 through 1999 in the SENTRY Antimicrobial Surveillance Program. J Clin Microbiol 39, 3254–3259.[CrossRef]
    [Google Scholar]
  28. Playford, E. G., Kong, F., Sun, Y., Wang, H., Halliday, C. & Sorrell, T. C. ( 2006; ). Simultaneous detection and identification of Candida, Aspergillus, and Cryptococcus species by reverse line blot hybridization. J Clin Microbiol 44, 876–880.[CrossRef]
    [Google Scholar]
  29. Rex, J. H., Bennett, J. E., Sugar, A. M., Pappas, P. G., van der Horst, C. M., Edwards, J. E., Washburn, R. G., Scheld, W. M., Karchmer, A. W. & other authors ( 1994; ). A randomized trial comparing fluconazole with amphotericin B for the treatment of candidaemia in patients without neutropenia. N Engl J Med 331, 1325–1330.[CrossRef]
    [Google Scholar]
  30. Rigby, S., Procop, G. W., Haase, G., Wilson, D., Hall, G., Kurtzman, C., Oliveira, K., Von Oy, S., Hyldig-Nielsen, J. J. & other authors ( 2002; ). Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J Clin Microbiol 40, 2182–2186.[CrossRef]
    [Google Scholar]
  31. Selvarangan, R., Bui, U., Limaye, A. P. & Cookson, B. T. ( 2003; ). Rapid identification of commonly encountered Candida species directly from blood culture bottles. J Clin Microbiol 41, 5660–5664.[CrossRef]
    [Google Scholar]
  32. Shin, J. H., Nolte, F. S. & Morrison, C. J. ( 1997; ). Rapid identification of Candida species in blood cultures by a clinically useful PCR method. J Clin Microbiol 35, 1454–1459.
    [Google Scholar]
  33. 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.
    [Google Scholar]
  34. Trick, W. E., Fridkin, S. K., Edwards, J. R., Hajjeh, R. A. & Gaynes, R. P. ( 2002; ). Secular trend of hospital-acquired candidaemia among intensive care unit patients in the United States during 1989–1999. Clin Infect Dis 35, 627–630.[CrossRef]
    [Google Scholar]
  35. Wey, S. B., Mori, M., Pfaller, M. A., Woolson, R. F. & Wenzel, R. P. ( 1988; ). Hospital-acquired candidemia. The attributable mortality and excess length of stay. Arch Intern Med 148, 2642–2645.[CrossRef]
    [Google Scholar]
  36. White, P. L., Shetty, A. & Barnes, R. A. ( 2003; ). Detection of seven Candida species using the Light-Cycler system. J Med Microbiol 52, 229–238.[CrossRef]
    [Google Scholar]
  37. Wilson, D. A., Joyce, M. J., Hall, L. S., Reller, L. B., Roberts, G. D., Hall, G. S., Alexander, B. D. & Procop, G. W. ( 2005; ). Multicenter evaluation of a Candida albicans peptide nucleic acid fluorescent in situ hybridization probe for characterization of yeast isolates from blood cultures. J Clin Microbiol 43, 2909–2912.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.47149-0
Loading
/content/journal/jmm/10.1099/jmm.0.47149-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error