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Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance
Published on: December 28, 2017
Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus
Sara Gago1,2, Carmen Serrano3, Ana Alastruey-Izquierdo1,4
1Mycology Reference Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
Abstract:
Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA4 microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patient's management.
Insights
Cryptococcus deneoformans and hybrids are common in HIV/AIDS patients with meningitis. Molecular characterization and antifungal susceptibility are crucial for managing cryptococcal meningitis, especially with emerging fluconazole resistance.
Area of Science:
- Mycology
- Infectious Diseases
- Molecular Biology
Background:
- Cryptococcal meningitis is a major cause of mortality in individuals with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS).
- Understanding the diversity, antifungal resistance, and virulence of Cryptococcus species is critical for effective patient management.
Purpose of the Study:
- To characterize the epidemiology, antifungal susceptibility, and virulence of Cryptococcus species isolated from HIV/AIDS patients.
- To investigate the molecular mechanisms of fluconazole resistance in clinical Cryptococcus strains.
Main Methods:
- Molecular characterization including serotyping, mating type determination, PCR fingerprinting (M13, GACA4), and rDNA region analysis (IGS1, ITS).
- Antifungal susceptibility testing following CLSI protocols and ERG11 gene sequencing for resistance mechanisms.
- Virulence assessment using a Galleria mellonella infection model.
Main Results:
- Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids constituted 65% of isolates, with C. neoformans comprising 35%.
- Thirteen distinct microsatellite genotypes were identified, and mixed infections were observed in three patients.
- Twenty-nine percent of strains exhibited fluconazole resistance, with one case linked to an ERG11 gene mutation (G470R).
- C. neoformans strains demonstrated higher virulence in the G. mellonella model compared to C. deneoformans and hybrids.
Conclusions:
- Cryptococcus deneoformans and its hybrids are prevalent in HIV/AIDS patients with meningitis in this Spanish cohort.
- Molecular identification and antifungal susceptibility testing are essential for guiding treatment decisions, particularly given the prevalence of fluconazole resistance.
- Understanding Cryptococcus species-specific virulence is important for predicting disease severity and outcomes in immunocompromised patients.
