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A Rapid, Visible, and Highly Sensitive Method for Recognizing and Distinguishing Invasive Fungal Infections via
Qiong Yang1, Binghong He1, Chong Chen2
1Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing 100875, China.
Abstract:
Invasive fungal infection (IFI) is one of the leading causes of death in the intensive care unit (ICU) due to its high morbidity and mortality among immunocompromised patients. Early diagnosis of IFI is typically infeasible because of the lack of clinical signs and symptoms. By virtue of the cationic conjugated polymer-based fluorescence resonance energy transfer (CCP-FRET) technology, we develop a rapid, visible, simple, and sensitive method for simultaneous detection and discrimination of three types of pathogens, including Candida albicans (C. albicans), Klebsiella pneumoniae (K. pneumoniae), and Cryptococcus neoformans (C. neoformans). The CCP-FRET system contains a CCP fluorescent probe and pathogen-specific DNA labeled with fluorescent dyes. These two components spontaneously self-assemble into the complex under electrostatic attraction, resulting in an efficient FRET from CCP to fluorescent dyes when irradiated with a 380 nm ultraviolet (UV) light. The CCP-FRET method can specifically identify the DNA molecules that are extracted from culture pathogen strains or blood samples via PCR and single base extension (SBE) reactions, without any cross-reactions on the DNA of nonspecific strains. In particular, the sensitivity of this method is down to 0.03125 ng, which is ten times higher than that of real-time PCR. We further evaluate its detection efficiency by testing 15 blood samples from neonatal patients who suffer from pathogen infections, in which some of them have undergone antipathogen treatments. Using the CCP-FRET method, 33.3% (5/15) of samples tested positive for C. albicans and/or K. pneumoniae infections, whereas no pathogen DNAs are recognized with real-time PCR, despite using the same primers. Interestingly, the CCP-FRET method can output unique fluorescent color as well as RGB patterns to different types of pathogen infections, by which the infection type can be conveniently determined. Collectively, the CCP-FRET method is a sensitive and reliable detection platform for rapid identification of fungal and bacterial multiple infections, holding great promise for uses in clinical testing.
Insights
A new cationic conjugated polymer-based fluorescence resonance energy transfer (CCP-FRET) method enables rapid, sensitive detection of invasive fungal infections (IFIs). This technology offers a promising tool for early diagnosis and discrimination of multiple pathogens in clinical settings.
Area of Science:
- Biotechnology and Biomedical Engineering
- Molecular Diagnostics
- Infectious Disease Detection
Background:
- Invasive fungal infections (IFIs) are a major cause of mortality in intensive care units (ICUs), particularly among immunocompromised patients.
- Early diagnosis of IFIs is challenging due to the lack of specific clinical signs and symptoms.
- Current diagnostic methods may lack the sensitivity and speed required for timely intervention.
Purpose of the Study:
- To develop a rapid, visible, simple, and sensitive method for simultaneous detection and discrimination of key pathogens causing IFIs.
- To utilize cationic conjugated polymer-based fluorescence resonance energy transfer (CCP-FRET) technology for enhanced pathogen identification.
- To evaluate the clinical utility of the CCP-FRET method in identifying fungal and bacterial infections from patient samples.
Main Methods:
- Development of a CCP-FRET system comprising a CCP fluorescent probe and pathogen-specific DNA labeled with fluorescent dyes.
- Utilizing electrostatic attraction for spontaneous self-assembly of the CCP-FRET complex.
- Detection of pathogen DNA from cultured strains or blood samples via PCR and single-base extension (SBE) reactions, with detection via UV light irradiation.
Main Results:
- The CCP-FRET method achieved high specificity, detecting DNA from *Candida albicans*, *Klebsiella pneumoniae*, and *Cryptococcus neoformans* without cross-reactions.
- Demonstrated a sensitivity of 0.03125 ng, significantly higher (tenfold) than real-time PCR.
- Identified infections in 33.3% of neonatal patient blood samples, including cases missed by real-time PCR, and provided unique fluorescent color/RGB patterns for pathogen discrimination.
Conclusions:
- The CCP-FRET method is a sensitive, reliable, and rapid platform for identifying multiple fungal and bacterial infections.
- This technology holds significant promise for improving clinical diagnostic capabilities for invasive infections.
- The distinct fluorescent outputs facilitate convenient determination of infection types.
