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Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples
Raquel Sabino1, Helena Simões1, Cristina Veríssimo1
1Reference Unit for Parasitic and Fungal Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal.
Abstract:
Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients' comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we to present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected.
Insights
Real-time multiplex PCR aids in diagnosing invasive aspergillosis by detecting Aspergillus DNA in tissue samples. This molecular method proved useful, especially when cultures were negative, identifying Aspergillus fumigatus but not azole resistance mutations.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Diagnosing invasive fungal infections, particularly invasive aspergillosis, presents challenges due to method variability.
- Standardization of molecular diagnostic techniques for fungal infections remains an unmet need.
- Nucleic acid testing offers advantages over antigen assays by identifying species and detecting resistance mutations.
Purpose of the Study:
- To evaluate the utility of a real-time multiplex PCR assay for diagnosing aspergillosis in tissue samples.
- To demonstrate the application of this PCR method in identifying Aspergillus species and detecting azole resistance.
- To present laboratory cases showcasing the diagnostic value of Aspergillus real-time multiplex PCR.
Main Methods:
- Real-time multiplex PCR was employed for the direct detection of Aspergillus DNA in patient tissue samples.
- Panfungal PCR and sequencing were used to confirm positive Aspergillus PCR results.
- The assay was also utilized to screen for mutations associated with azole resistance.
Main Results:
- The real-time multiplex PCR assay successfully detected Aspergillus DNA in tissue samples, aiding in aspergillosis diagnosis.
- In most PCR-positive cases, fungal cultures remained negative after 60 days of incubation.
- Positive PCR signals identified Aspergillus fumigatus sensu stricto, confirmed by sequencing. No azole resistance mutations were detected.
Conclusions:
- Real-time multiplex PCR serves as a valuable tool for diagnosing aspergillosis directly from tissue samples.
- The molecular assay demonstrated effectiveness in identifying Aspergillus fumigatus, even when traditional cultures were uninformative.
- Further investigation is warranted to fully assess the capability of detecting azole resistance using this PCR approach.
