Rapid duplex flap probe-based isothermal assay to identify the Cryptococcus neoformans and Cryptococcus gattii

Area of Science:

• Mycology
• Molecular Diagnostics
• Infectious Diseases

Background:

• Cryptococcosis, caused by *Cryptococcus neoformans* and *Cryptococcus gattii*, presents increasing global mortality.
• Accurate differentiation between these species is crucial due to distinct clinical and epidemiological profiles.
• Current diagnostic methods like culture and antigen detection lack the specificity for differential diagnosis.

Purpose of the Study:

• To develop a rapid and accurate diagnostic assay for differentiating *Cryptococcus neoformans* and *Cryptococcus gattii*.
• To provide a tool for timely and precise differential diagnosis of cryptococcosis.

Main Methods:

• Established a novel duplex flap probe-based isothermal assay.
• Combined nucleic acid isothermal amplification with fluorescence probe technology.
• Utilized two distinct fluorescence flap probes in a single reaction system to detect sequence variations.

Main Results:

• The assay achieved identification of *Cryptococcus neoformans* and *Cryptococcus gattii* within 1 hour.
• Demonstrated high sensitivity (10 copies/μL) and specificity (100%) compared to traditional methods.
• Validated performance in spiked clinical samples, including cerebrospinal fluid and bronchoalveolar lavage fluid.

Conclusions:

• The developed duplex flap probe-based isothermal assay is a robust and rapid tool for differential diagnosis of cryptococcosis.
• This assay shows significant promise for clinical settings, particularly when cryptococcal disease is suspected.
• Facilitates improved patient management and epidemiological studies through accurate species identification.