1887

Abstract

This study describes the development of a novel assay to detect fungal DNA and identify the most clinically relevant invasive human pathogenic fungi to the species level using oligonucleotide probes, labelled with electrochemically active groups, and solid-state electrodes. A panfungal probe designed against the 18S rRNA gene region, capable of detecting all fungal pathogens tested, and species-specific probes, designed against the ITS2 region for detection of the five species most commonly encountered in the clinical setting (, , species complex, and ), are described. When tested with PCR-amplified DNA from both type and clinical strains of the relevant species, the probes were able to positively identify the relevant fungi, indicated by production of a current significantly elevated above the background reading. No cross-reactivity was observed with any of the species-specific probes when compared with nine non-target species or in the presence of human DNA equivalent to an equal number of ITS2 targets. The panfungal probe gave results that were similarly positive against 15 other fungal species and also did not cross-react with human DNA. The limit of detection of the assay was shown to be approximately 1 genome equivalent for all probes using extracted genomic DNA.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.009183-0
2009-09-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/58/9/1182.html?itemId=/content/journal/jmm/10.1099/jmm.0.009183-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
    [Google Scholar]
  2. Arendrup M. C., Fuursted K., Gahrn-Hansen B., Schønheyder H. C., Knudsen J. D., Jensen I. M., Bruun B., Christensen J. J., Johansen H. K. 2008; Semi-national surveillance of fungaemia in Denmark 2004–2006: increasing incidence of fungaemia and numbers of isolates with reduced azole susceptibility. Clin Microbiol Infect 14:487–494 [CrossRef]
    [Google Scholar]
  3. Badiee P., Kordbacheh P., Alborzi A., Malekhoseini S., Ramzi M., Mirhendi H., Mahmoodi M., Shakiba E. 2009; Study on invasive fungal infections in immunocompromised patients to present a suitable early diagnostic procedure. Int J Infect Dis 13:97–102 [CrossRef]
    [Google Scholar]
  4. Baskova L., Landlinger C., Preuner S., Lion T. 2007; The Pan-AC assay: a single-reaction real-time PCR test for quantitative detection of a broad range of Aspergillus and Candida species. J Med Microbiol 56:1167–1173 [CrossRef]
    [Google Scholar]
  5. Clancy C. J., Nguyen M. L., Cheng S., Huang H., Fan G., Jaber R. A., Wingard J. R., Cline C., Nguyen M. H. 2008; Immunoglobulin G responses to a panel of Candida albicans antigens as accurate and early markers for the presence of systemic candidiasis. J Clin Microbiol 46:1647–1654 [CrossRef]
    [Google Scholar]
  6. 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]
  7. 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]
  8. Hajjeh R. A., Sofair A. N., Harrison L. H., Lyon G. M., Arthington-Skaggs B. A., Mirza S. A., Phelan M., Morgan J., Lee-Yang W. other authors 2004; Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol 42:1519–1527 [CrossRef]
    [Google Scholar]
  9. Hata D. J., Buckwalter S. P., Pritt B. S., Roberts G. D., Wengenack N. L. 2008; Real-time PCR method for detection of zygomycetes. J Clin Microbiol 46:2353–2358 [CrossRef]
    [Google Scholar]
  10. Hillier S. C., Flower S. E., Frost C. G., Jenkins A. T. A., Keay R., Braven H., Flower S. E., Clarkson J. M. 2004; An electrochemical gene detection assay utilising T7 exonuclease activity on complementary probe-target oligonucleotide sequences. Electrochem Commun 6:1227–1232 [CrossRef]
    [Google Scholar]
  11. Horn D., Neofytos D., Fishman J., Steinbach W., Anaisie E., Marr K. A., Pfaller M., Olyaei A. 2007; Use of the PATH Alliance database to measure adherence to IDSA guidelines for the therapy of candidemia. Eur J Clin Microbiol Infect Dis 26:907–914 [CrossRef]
    [Google Scholar]
  12. Innings A., Ullberg M., Johansson A., Rubin C. J., Noreus N., Isaksson M., Herrmann B. 2007; Multiplex real-time PCR targeting the RNase P RNA gene for detection and identification of Candida species in blood. J Clin Microbiol 45:874–880 [CrossRef]
    [Google Scholar]
  13. Kao A. S., Brandt M. E., Pruitt W. R., Conn L. A., Perkins B. A., Stephens D. S., Baughman W. S., Reingold A. L., Rothrock G. A. other authors 1999; The epidemiology of candidemia in two United States cities: results of a population-based active surveillance. Clin Infect Dis 29:1164–1170 [CrossRef]
    [Google Scholar]
  14. Kerr C., Sadowski P. D. 1972; Gene 6 exonuclease of bacteriophage T7. II. Mechanism of the reaction. J Biol Chem 247:311–318
    [Google Scholar]
  15. Kullman B., Tamm H., Kullman K. 2005; Fungal genome size database. Accessed 15/03/2009 http://www.zbi.ee/fungal-genomesize
    [Google Scholar]
  16. Lau A., Sorrell T. C., Chen S., Stanley K., Iredell J., Halliday C. 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 [CrossRef]
    [Google Scholar]
  17. Linton C. J., Borman A. M., Cheung G., Holmes A. D., Szekely A., Palmer M. D., Bridge P. D., Campbell C. K., Johnson E. M. 2007; Molecular identification of unusual pathogenic yeast isolates by large ribosomal subunit gene sequencing: 2 years of experience at the United Kingdom Mycology Reference Laboratory. J Clin Microbiol 45:1152–1158 [CrossRef]
    [Google Scholar]
  18. 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]
  19. Metwally L., Hogg G., Coyle P. V., Hay R. J., Hedderwick S., McCloskey B., O'Neill H. J., Ong G. M., Thompson G. other authors 2007; Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR. J Med Microbiol 56:964–970 [CrossRef]
    [Google Scholar]
  20. Pfaller M. A., Diekema D. J., Gibbs D. L., Newell V. A., Bijie H., Dzierzanowska D., Klimko N. N., Letscher-Bru V., Lisalova M. other authors 2009; Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of noncandidal yeast species to fluconazole and voriconazole determined by CLSI standardized disk diffusion testing. J Clin Microbiol 47:117–123 [CrossRef]
    [Google Scholar]
  21. 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]
  22. 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]
  23. 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 [CrossRef]
    [Google Scholar]
  24. Tortorano A. M., Peman J., Bernhardt H., Klingspor L., Kibbler C. C., Faure O., Biraghi E., Canton E., Zimmermann K. other authors 2004; Epidemiology of candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study. Eur J Clin Microbiol Infect Dis 23:317–322 [CrossRef]
    [Google Scholar]
  25. van Deventer A. J., Goessens W. H., van Belkum A., van Vliet H. J., van Etten E. W., Verbrugh H. A. 1995; Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis. J Clin Microbiol 33:625–628
    [Google Scholar]
  26. Vollmer T., Störmer M., Kleesiek K., Dreier J. 2008; Evaluation of novel broad-range real-time PCR assay for rapid detection of human pathogenic fungi in various clinical specimens. J Clin Microbiol 46:1919–1926 [CrossRef]
    [Google Scholar]
  27. White T. J., Burns T. D., Lee S. B., Taylor J. W. 1990; Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications . pp 315–322 Edited by Innis M. A., Gelfand D. H., Sninsky J. J., White T. J. San Diego, CA: Academic Press;
  28. Zhao Y., Park S., Kreiswirth B. N., Ginnochio C. C., Veyret R., Laayoun A., Troesch A., Perlin D. S. 2009; A rapid real-time nucleic acid sequence-based amplification (NASBA)-molecular beacons platform to detect fungal and bacterial bloodstream infections. J Clin Microbiol (in press
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.009183-0
Loading
/content/journal/jmm/10.1099/jmm.0.009183-0
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