Detection and identification of Aspergillus spp. and Candida spp. by real-time PCR in clinical samples
Abstract number: 1733_1361
Torres M.J., Aller A., Ramírez M., Castro C., Ruiz M., Cisneros J.M., Espigado I., Aznar J., Martín-Mazuelos E., Palomares J.C.
Objective: To develop and evaluate prospectively a real-time PCR assay for detection and identification of Aspergillus spp. and Candida spp. in patients with suspected IFI.
Methods: Referenced cultures of Aspergillus species and Candida species were included to determine the analytical sensitivity and specificity of the assay.
The amplification method used the 18S rRNA genes and the identification of the genus was made by two specific pair of probes. The analytical sensitivity of the process was evaluated with suspensions of A. fumigatus and Candida albicans in five concentrations (101 to 105 cfu. per ml), and serially diluted DNA of the referred species.
Total genomic DNA extracted from 948 blood samples obtained from 127 patients suspected of having IFI were used.
Results: No cross-reactivity was obtained with any of the collection of pathogenic and non-pathogenic bacteria and fungi used in the study.
Species identification was determined by analysing the Tm of the melting curves obtained with the specific probes, ranging from 67.34°C to 70.7°C for Aspergillus spp. and from 51.3°C to 64.5°C for Candida spp. Analytical sensitivity for Aspergillus fumigatus was 60 fg using DNA and 15 conidia using conidial suspensions; and 100 fg and 3 cells for Candida albicans.
There were 2 cases of proven IA, 3 cases of probable IA and 14 cases of possible IA. One patient was classified as having probable Candidiasis, and 6 cases possible Candidiasis. Only patients with serial positive results were considered to be PCR positive.
The 2 IA proven cases and 2 of 3 IA probable cases were PCR positive for Aspergillus and all the cases with probable/possible Candidiasis had positive PCR results for Candida albicans.
Conclusions: The Light Cycler technique is rapid, accurate, and reproducible and combines rapid in vitro amplification with real-time species determination and quantification of the fungal load. The linear range of the assay was from 6 to 6×107 fg of Aspergillus DNA and from 10 to 107 fg of Candida DNA. The assay used provides high sensitivity and specificity for fungal detection but more cases are needed to elucidate the true potential of the technique.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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