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Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants
Published on: May 5, 2023
An overview of using fungal DNA for the diagnosis of invasive mycoses
P Lewis White1, Alexandre Alanio2,3,4, Lottie Brown5
1Public Health Wales Microbiology Cardiff, UHW, Cardiff, UK.
Introduction:
Fungal PCR has undergone considerable standardization and, together with the availability of commercial assays, external quality assessment schemes, and extensive performance validation data, is ready for widespread use for the screening and diagnosis of invasive fungal disease (IFD).
Areas Covered:
Drawing on the experience and knowledge of the leads of the various working parties of the Fungal PCR initiative, this review will address general considerations concerning the use of molecular tests for the diagnosis of IFD, before focusing specifically on the technical and clinical aspects of molecular testing for the main causes of IFD and recent technological developments.
Expert Opinion:
For infections caused by Aspergillus, Candida, and Pneumocystis jirovecii, PCR testing is recommended, and combination with serological testing will likely enhance the diagnosis. For other IFD (e.g. mucormycosis), molecular diagnostics represent the only non-classical mycological approach toward diagnoses, and continued performance validation and standardization have improved confidence in such testing. The emergence of antifungal resistance can be diagnosed, in part, through molecular testing. Next-generation sequencing has the potential to significantly improve our understanding of fungal phylogeny, epidemiology, pathogenesis, mycobiome/microbiome, and interactions with the host, while identifying novel and existing mechanisms of antifungal resistance and novel diagnostic/therapeutic targets.
Insights
Molecular PCR testing is now ready for diagnosing invasive fungal diseases (IFD). This review covers its use, focusing on key fungi and emerging technologies like next-generation sequencing for improved diagnostics and resistance detection.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Standardization of fungal PCR assays and availability of commercial tests facilitate widespread use for invasive fungal disease (IFD) diagnosis.
- External quality assessment schemes and validation data support the readiness of PCR for clinical application.
Purpose of the Study:
- To review general considerations for molecular tests in IFD diagnosis.
- To focus on technical and clinical aspects of molecular testing for major IFD pathogens.
- To discuss recent technological advancements in fungal diagnostics.
Main Methods:
- Expert review drawing on working party knowledge.
- Focus on established and emerging molecular diagnostic techniques for IFD.
- Discussion of clinical utility and validation of molecular assays.
Main Results:
- PCR testing is recommended for infections caused by Aspergillus, Candida, and Pneumocystis jirovecii, often enhanced by serological tests.
- Molecular diagnostics offer the sole non-classical mycological approach for certain IFDs like mucormycosis.
- Molecular testing aids in diagnosing antifungal resistance and identifying novel diagnostic/therapeutic targets.
Conclusions:
- Molecular diagnostics, including PCR, are crucial for IFD diagnosis, especially for difficult-to-culture fungi.
- Next-generation sequencing holds significant potential for advancing fungal research, diagnostics, and understanding antifungal resistance.
- Continued validation and standardization enhance confidence in molecular testing for IFD.
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