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Updated: Aug 20, 2026
Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body
Published on: January 11, 2018
Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene
Muhammad Amjad1, Najla Kfoury1, Raymond Cha1
1Clinical Laboratory Science Program1 and Department of Pharmacy Practice2, Eugene Applebaum College of Pharmacy and Health Services, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, USA 3,4Medical Research Program3 and School of Medicine4, Wayne State University, 540 E. Canfield Street, Detroit, MI 48201, USA.
Abstract:
Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1-10 c.f.u. ml(-1) of the sample. No amplification was observed from up to 10(5) heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.
Insights
A new LightCycler RT-PCR method accurately detects viable Cryptococcus neoformans by quantifying its capsule gene mRNA. This sensitive assay aids in diagnosing cryptococcal meningitis and evaluating antifungal treatment efficacy.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Cryptococcus neoformans is an opportunistic fungal pathogen causing life-threatening meningitis, particularly in immunocompromised individuals.
- Accurate and rapid detection of viable C. neoformans is crucial for timely treatment and monitoring therapeutic response.
Purpose of the Study:
- To establish a quantitative and viability-assessment method for C. neoformans using LightCycler RT-PCR.
- To develop a species-specific assay for detecting viable C. neoformans in clinical specimens.
Main Methods:
- LightCycler RT-PCR amplification targeting the CAP10 gene mRNA of C. neoformans.
- Development of an internal control using in vitro-transcribed RNA.
- Sensitivity determination using serial dilutions of RNA and C. neoformans cultures.
Main Results:
- The assay demonstrated high specificity, detecting as little as 0.472 fg of capsule mRNA, corresponding to 1-10 c.f.u./mL.
- No cross-amplification was observed with non-viable yeast cells or other microbial nucleic acids.
- A single band of 223 bp was consistently observed via agarose gel electrophoresis.
Conclusions:
- LightCycler RT-PCR amplification of capsule mRNA is a sensitive, specific, and quantitative method for detecting viable C. neoformans.
- This technique can significantly aid in diagnosing cryptococcal meningitis and assessing antifungal drug efficacy.
