HTS and PCR Methods Are the Most Used in the Diagnosis of Aspergillosis: Advantages over Other Molecular Methods

Area of Science:

• Medical Mycology
• Molecular Diagnostics
• Infectious Diseases

Background:

• Aspergillosis encompasses a spectrum of diseases caused by Aspergillus species.
• Invasive aspergillosis poses a life-threatening risk, particularly to immunocompromised individuals.
• Improved diagnostic tools are essential for effective patient management.

Purpose of the Study:

• To review and compare commonly used molecular diagnostic methods for aspergillosis.
• To emphasize the advantages of various molecular techniques for clinical sample analysis.
• To identify the most sensitive and specific methods for aspergillosis detection.

Main Methods:

• High-Throughput Sequencing (HTS)
• Next-Generation Sequencing (NGS)
• In Situ Hybridization (ISH)
• Microarrays
• PCR-Restriction Fragment Length Polymorphism (PCR-RFLP)
• Loop-mediated Isothermal Amplification (LAMP)
• Polymerase Chain Reaction (PCR) variants (qPCR, multiplex PCR, nested PCR, RT-PCR, endpoint PCR, U-dHRM, ddPCR)

Main Results:

• Next-Generation Sequencing (NGS) and various Polymerase Chain Reaction (PCR) modalities are the most frequently employed methods.
• Quantitative PCR (qPCR) exhibits superior sensitivity and specificity across diverse clinical samples (blood, serum, BAL, tissue, sputum).
• Next-Generation Sequencing (NGS) identifies bronchoalveolar lavage (BAL) as the optimal sample type, yielding higher fungal loads compared to sputum or blood.

Conclusions:

• Quantitative PCR (qPCR) demonstrates significant progress in sensitivity and specificity for diagnosing aspergillosis.
• Next-Generation Sequencing (NGS) is a powerful, non-targeted technique, but its sensitivity can be reduced by high background nucleic acids in clinical samples.
• The choice of molecular method and clinical sample type impacts diagnostic accuracy for aspergillosis.