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Touchdown PCR as a tool for improved detection of invasive candidosis

Abstract number: 1734_98

Angelov P., Kantardjiev T., Levterova V., Zamfirova E., Lesseva M., Vacheva R., Bobcheva S., Shopova E.

Summary: A touchdown conventional PCR based on the internal transcribed spacer sequence (ITS1) was developed for the detection of Candida albicans and five other Candida species. The assay precisely identifies C. glabrata, C. parapsilosis, C. lusitaniae, C. guilliermondii, and C. krusei, which are clinically most frequent causative agents of systemic infections. An evaluation of the assay with artificially prepared samples revealed a detection limit of 2–4 genomic copies for C. albicans and the other species.

Background: Invasive candidosis poses a major cause of morbidity and mortality in severe immunocompromised and hospitalised patients. Regarding the high mortality rates, there is necessity for rapid and accurate diagnostic methods for rapid diagnostics. The internal transcribed spacer sequences (ITS) are localised between the genes for small subunit r RNA and 5.8S r RNA, and large subunit r RNA, which are conserved among all fungi, but vary among different species, posing suitable target for diagnostic aims.

Aims: To study the ability of touchdown PCR to improve the detection of invasive candidosis and species identification of the pathogen.

Methods: Reaction conditions were optimised, using referent Candida strains DNA, and the product was visualised on ethidium bromide-stained agarose gel electrophoresis.

Serum samples from 11 patients with proven invasive candidosis were obtained, and 200 microliters from each sample were subjected to DNA extraction using DNA Purification Kit® (Fermentas®).

Touchdown PCR-assay was developed, using universal fungal primers ITS1 and ITS2 (1), which targets ITS1 region. 40 cycles of the reaction was performed, at annealing temperature ranging 68–58 degrees for the first 10 cycles, and being constant during the next 30 cycles.

Results: The assay reached sensitivity equal to 2–4 C. albicans genomes, allowing identification of six Candida species.

The touchdown PCR was able to detect fungal DNA in all tested specimens, and C. albicans, C. krusei, C. parapsilosis and C. glabrata were identified, proving the identification results, achieved with the blood culture system Bac T/ALERT FA®.

Conclusion: A sensitive and specific touchdown PCR-based assay was developed for the detection of six Candida species in serum specimens. Four of the most relevant pathogenic species were successfully identified in serum specimens from patients with sepsis.

References

[1] Hibbett D. Ribosomal RNA and fungal systematics. Trans Mycol Soc Jpn. 1992; 33: 533–556.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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