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Published on: December 28, 2017
Molecular genotyping of Candida parapsilosis group I clinical isolates by analysis of polymorphic microsatellite
Brent A Lasker1, Geraldine Butler, Timothy J Lott
1Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Centers for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Mailstop G-11, Atlanta, GA 30333. blasker@cdc.gov
Abstract:
Candida parapsilosis, a pathogenic yeast, is composed of three newly designated genomic species that are physiologically and morphologically indistinguishable. Nosocomial infections caused by group I C. parapsilosis are often associated with the breakdown of infection control practices and the contamination of medical devices, solutions, and indwelling catheters. Due to the low levels of nucleotide sequence variation that are observed, an investigation of the size polymorphisms in loci harboring microsatellite repeat sequences was applied for the typing of C. parapsilosis group I isolates. PCR primer sets that flank the microsatellite repeats for seven loci were designed. Following amplification by PCR, the size of each amplification product was determined automatically by capillary electrophoresis. A total of 42 C. parapsilosis group I isolates were typed by microsatellite analysis, and their profiles were compared to the hybridization profiles obtained by use of the Cp3-13 DNA probe. A high degree of discrimination (discriminatory power = 0.971) was observed by microsatellite analysis. The number of different alleles per locus ranged from 14 for locus B to 5 for locus C. Microsatellite analysis detected 30 different microsatellite genotypes, with 24 genotypes represented by a single isolate. Comparison of the genotypes obtained by microsatellite analysis and those obtained by analysis of the Cp3-13 hybridization profiles showed that they were similar, and these methods were able to identify related and unrelated isolates. Some discrepancies were observed between the methods and may be due to higher mutation rates and/or homoplasy by microsatellite markers. Identical results were observed between microsatellite analysis and Cp3-13 DNA hybridization profile analysis for C. parapsilosis isolates obtained from two patients, demonstrating the reproducibilities of the methods in vivo. Identical microsatellite profiles were observed for isolates displaying different phenotypic switching morphologies. Indistinguishable Cp3-13 DNA hybridization profiles were observed for six epidemiologically related isolates; however, only three of six primary isolates had identical microsatellite profiles. Size variation at a single locus was observed for three of six isolates obtained either after the outbreak period or from a different body site, suggesting the potential of the method to detect microevolutionary events. Interestingly, for most loci a single allele per strain was observed; in contrast, two alleles per locus were observed for some strains, and consistent with the findings for natural isolates, some isolates may be aneuploid. Due to the potential for high throughput, reproducibility, and discrimination, microsatellite analysis may provide a robust and efficient method for the genotyping of large numbers of C. parapsilosis group I isolates.
Insights
Microsatellite analysis offers a highly discriminatory method for identifying genetic variations in Candida parapsilosis group I isolates. This technique provides a robust and efficient approach for genotyping, aiding in the understanding of nosocomial infections.
Area of Science:
- Medical Mycology
- Molecular Epidemiology
- Infectious Diseases
Background:
- Candida parapsilosis group I causes nosocomial infections, often linked to contaminated medical devices and poor infection control.
- Low nucleotide sequence variation in C. parapsilosis group I necessitates alternative typing methods.
- Understanding the genetic diversity of C. parapsilosis is crucial for effective infection control and outbreak management.
Purpose of the Study:
- To evaluate the utility of microsatellite analysis for typing Candida parapsilosis group I isolates.
- To compare the discriminatory power of microsatellite analysis with existing DNA fingerprinting methods (Cp3-13 probe).
- To assess the reproducibility and potential for detecting microevolutionary events using microsatellite markers.
Main Methods:
- Design of PCR primer sets flanking microsatellite repeat sequences in seven loci.
- Amplification of microsatellites by PCR, followed by size determination using capillary electrophoresis.
- Typing of 42 C. parapsilosis group I isolates and comparison with Cp3-13 DNA hybridization profiles.
Main Results:
- Microsatellite analysis demonstrated high discriminatory power (0.971), identifying 30 distinct genotypes among 42 isolates.
- The method revealed significant genetic diversity, with allele numbers ranging from 5 to 14 per locus.
- Microsatellite analysis showed good concordance with Cp3-13 hybridization but offered superior discrimination, detecting microevolutionary changes and potential aneuploidy.
Conclusions:
- Microsatellite analysis is a robust, reproducible, and highly discriminatory method for genotyping Candida parapsilosis group I.
- This technique is suitable for high-throughput analysis, aiding in epidemiological surveillance and infection control.
- Microsatellite analysis can potentially detect microevolutionary events and genetic variations like aneuploidy in C. parapsilosis.
