What is a pan fungal DNA (Deoxyribonucleic Acid) test?

Pan Fungal DNA Test: A Molecular Diagnostic Tool for Fungal Infections

A pan fungal DNA test is a molecular diagnostic method that detects fungal DNA from a wide range of fungal species in clinical specimens, allowing for broad identification of fungal pathogens when the specific causative organism is unknown. 1

Mechanism and Methodology

• Pan fungal PCR assays typically target conserved fungal DNA regions such as the 18S rRNA subunit gene, 28S rRNA gene, or internal transcribed spacer (ITS) regions that are present in almost all fungal species 1
• These tests employ primers that bind to these conserved regions, followed by DNA amplification and detection using various techniques including:
  • Conventional PCR followed by hybridization with species-specific probes 1
  • Real-time PCR technology (e.g., LightCycler, TaqMan) which allows for potential quantification of fungal burden 1
  • DNA microarray hybridization for simultaneous detection of multiple fungal species 2

Clinical Applications

• Particularly valuable in immunocompromised patients with suspected invasive fungal infections (IFIs) when the causative pathogen is unknown 1
• Can be performed on various clinical specimens including:
  • Bronchoalveolar lavage (BAL) fluid 3
  • Blood and serum samples 1
  • Tissue biopsies (fresh or formalin-fixed, paraffin-embedded) 4
  • Cerebrospinal fluid and other sterile body fluids 5
• Most useful when conventional methods (culture, histopathology) have failed to identify the causative pathogen 4

Performance Characteristics

• Sensitivity ranges from 65-100% depending on the specimen type, test methodology, and fungal burden 1, 5
• Specificity ranges from 65-75% for pan fungal PCR tests 1
• Testing BAL samples typically achieves higher sensitivity and specificity rates than testing blood samples 1
• High negative predictive value makes invasive disease less likely in patients with repeatedly negative PCR results 1

Advantages and Limitations

Advantages:

• Allows detection of fungal pathogens that are difficult to culture or when cultures remain negative 4
• Can detect fungal DNA even after antifungal treatment has been initiated 1
• Faster than traditional culture methods, potentially enabling earlier targeted therapy 6
• Can identify breakthrough infections or insufficient response to antifungal treatment 1

Limitations:

• Lack of standardization across laboratories 1
• Many tests require sequencing of PCR products for species identification, which increases turnaround time 6
• Potential for false positives due to environmental contamination 5
• Not yet included in mandatory recommendations for fungal diagnosis due to standardization issues 1

Current Recommendations

• The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and European Confederation of Medical Mycology (ECMM) moderately support the use of molecular methods on both fresh clinical material and paraffin slides for fungal diagnosis 1
• Fresh material is preferred over paraffin-embedded tissue because formalin damages DNA 1
• The American Thoracic Society recommends blood or serum Aspergillus PCR testing in severely immunocompromised patients suspected of having invasive pulmonary aspergillosis 1
• The German Society for Haematology and Oncology (AGIHO) acknowledges that molecular diagnostic tools are promising with high sensitivity and specificity, but notes they are not yet standardized or widely available 1

Best Practices

• Pan fungal DNA tests should be used in combination with other diagnostic methods such as antigen detection, culture, and histopathology for optimal diagnostic yield 1, 3
• Interpretation of results must be done in the context of clinical presentation and other laboratory findings 5
• Biopsy specimens for PCR testing should be diluted in isotonic saline rather than formalin to preserve DNA integrity 1
• Newer assays combining intercalating dyes with sequence-specific probes may allow for faster identification without the need for sequencing in many cases 6

Pan fungal DNA testing represents an important advancement in fungal diagnostics, particularly for difficult-to-diagnose cases, though continued standardization efforts are needed for broader clinical implementation.