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Updated: Aug 23, 2026
Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells
Published on: June 19, 2013
High-throughput detection of pathogenic yeasts of the genus trichosporon
1Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA. mdiaz@rsmas.miami.edu
Abstract:
The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10(2) genome molecules in PCRs and 10(7) to 10(8) molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.
Insights
A novel Luminex 100 flow cytometry method rapidly detects fungal pathogens like Trichosporon. This nucleotide hybridization assay offers high specificity and sensitivity for timely diagnosis of emerging fungal infections.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Flow Cytometry
Background:
- Increasing incidence of invasive fungal infections necessitates rapid and accurate diagnostic methods.
- The genus Trichosporon, a medically significant group of fungi, presents diagnostic challenges due to species-level similarities.
Purpose of the Study:
- To evaluate the Luminex 100 flow cytometer for the rapid and specific detection of Trichosporon species.
- To develop a multiplex nucleotide hybridization assay for fungal pathogen identification.
Main Methods:
- A nucleotide hybridization assay using fluorescent beads with species-specific probes targeting ribosomal DNA regions.
- Multiplex detection of 48 species-specific and group-specific probes.
- Quantification of hybridized amplicons using fluorescence detection.
Main Results:
- The assay demonstrated high specificity, distinguishing species with a single nucleotide difference.
- Rapid processing of a 96-well plate in under 50 minutes post-amplification.
- Sensitivity achieved detection of 10^2 genome molecules and 10^7-10^8 molecules of amplification product.
Conclusions:
- The Luminex 100 system provides a fast, specific, and sensitive method for Trichosporon species detection.
- This multiplex assay offers flexibility for identifying multiple fungal species simultaneously, aiding clinical diagnostics.
