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Identification of Aspergillus species using internal transcribed spacer regions 1 and 2
T Henry1, P C Iwen, S H Hinrichs
1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-6495, USA.
Abstract:
Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1-5.8S-ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA.
Insights
A new molecular method accurately identifies Aspergillus species, crucial for diagnosing invasive aspergillosis (IA) in immunocompromised patients. This technique aids in selecting effective antifungal treatments by distinguishing between species with varying virulence and drug resistance.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Aspergillus species are common causes of invasive mold infections, particularly in immunocompromised individuals.
- Accurate species identification is vital as different Aspergillus species exhibit varying virulence and antifungal resistance.
- Current identification methods may not always provide species-level resolution, impacting treatment decisions.
Purpose of the Study:
- To develop and validate a molecular method for accurate species-level identification of Aspergillus.
- To differentiate Aspergillus species from other pathogenic molds.
- To assess the clinical applicability of the developed method for diagnosing invasive aspergillosis.
Main Methods:
- Utilized 18S and 28S rRNA genes for primer design targeting the internal transcribed spacer (ITS) region (ITS 1-5.8S-ITS 2).
- Amplified, sequenced, and compared ITS regions from reference strains and clinical isolates of Aspergillus and other fungi.
- Performed a blind study on 11 clinical isolates to evaluate identification accuracy.
Main Results:
- The developed method successfully amplified ITS regions ranging from 565 to 613 bp for Aspergillus species.
- Both ITS 1 and ITS 2 regions were essential for accurate species-level identification.
- Minimal intraspecies variation was observed among clinical isolates and reference strains.
- Other pathogenic molds showed less than 89% sequence similarity to Aspergillus ITS regions.
- All 11 clinical isolates in the blind study were correctly identified.
Conclusions:
- The molecular method based on ITS sequencing provides accurate species-level identification of Aspergillus.
- This approach effectively differentiates Aspergillus from other molds, offering diagnostic value.
- Clinical application can lead to earlier diagnosis of invasive aspergillosis and informed selection of antifungal therapy.