Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay

Abstract

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi.

Identification of Medically Important Molds by an Oligonucleotide Array

“…The good performance (sensitivity and specificity) of the present array might be due to the fact that the fungal ITS sequences have low intraspecies variation and high interspecies divergence (10,19,21,24,32). Until now, there has been no report defining the criteria for maximum intraspecies variation in the ITS regions.…”

“…Invasive aspergillosis is a leading cause of infection among patients undergoing treatment for hematological malignancies, solid organ transplantation, and hematopoietic stem cell transplantation (8,10,37). Aspergillus fumigatus remains the most frequent cause of invasive aspergillosis, followed by A. flavus, A. terreus, A. niger, A. nidulans, and A. versicolor (12,22).…”

“…Although these published methods are useful for the identification of fungal species, they can identify only one or a limited number of species. The ribosomal internal transcribed spacer (ITS) regions also have been widely used as targets to detect and identify human fungal pathogens (5,7,10,21,23,24,(31)(32)(33)43).…”

Diagnosis of Invasive Fungal Infections – Current Limitations of Classical and New Diagnostic Methods

“…In general, assays targeting multicopy genes have better detection limits than those targeting single copy genes. 135 Although PCR may become a diagnostic modality to identify Mucorales, [136][137][138] Scedosporium, [139][140][141] and several other fungi, [142][143][144][145][146][147][148][149][150][151] no commercial test is available to date. These techniques hold promise, but they are not yet standardised or readily available in most clinical laboratories.…”

Investigating the Presence of Aspergillus fumigatus and A. flavus Using Galactomannan Enzyme Assay and TaqMan Real-Time PCR Technique

“…terreus is of concern. In addition, the probability to produce fulminant invasive infection in immunocompromised patients is possible (3). Numerous findings have indicated that a substantial cause of fungi found in hospitals is from potted plants (4).…”