Pyrosequencing Analysis of 20 Nucleotides of Internal Transcribed Spacer 2 Discriminates
 Candida parapsilosis
 ,
 Candida metapsilosis
 , and
 Candida orthopsilosis

Borman, Andrew M.; Linton, Christopher J.; Oliver, Debra; Palmer, M. Dean; Székely, Adrien; Odds, Frank C.; Johnson, Elizabeth

doi:10.1128/jcm.00240-09

Cited by 42 publications

(23 citation statements)

References 20 publications

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“…We have previously demonstrated that pyrosequencing of 35 bp of a portion of the internal transcribed spacer region 2 (ITS2) is sufficient to robustly identify the vast majority of pathogenic yeasts (27), including genetically closely related isolates within species complexes (28,29). This is also the case for the key species within the C. albicans species complex.…”

Section: Resultsmentioning

confidence: 93%

“…All 1,839 test isolates were subjected to pyrosequencing analysis of a portion of ITS2 exactly as described previously (27)(28)(29). For isolates identified by pyrosequencing as C. africana, identity was confirmed by PCR amplification and sequencing of a region of the large subunit gene (LSU) and the internal transcribed spacer 1 (ITS1) region using the primers described previously (30,31) and direct inoculation of PCR products with yeast suspensions as described in reference 32.…”

Section: Methodsmentioning

confidence: 99%

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Epidemiology, Antifungal Susceptibility, and Pathogenicity of Candida africana Isolates from the United Kingdom

Borman¹,

Székely²,

Linton³

et al. 2013

J Clin Microbiol

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Candida africana was previously proposed as a new species within the Candida albicans species complex, together with C. albicans and C. dubliniensis, although further phylogenetic analyses better support its status as an unusual variant within C. albicans. Here we show that C. africana can be distinguished from C. albicans and C. dubliniensis by pyrosequencing of a short region of ITS2, and we have evaluated its occurrence in clinical samples by pyrosequencing all presumptive isolates of C. albicans submitted to the Mycology Reference Laboratory over a 9-month period. The C. albicans complex constituted 826/1,839 (44.9%) of yeast isolates received over the study period and included 783 isolates of C. albicans, 28 isolates of C. dubliniensis, and 15 isolates of C. africana. In agreement with previous reports, C. africana was isolated exclusively from genital specimens, in women in the 18-to-35-year age group. Indeed, C. africana constituted 15/251 (6%) of "C. albicans" isolates from female genital specimens during the study period. C. africana isolates were germ tube positive, grew significantly more slowly than C. albicans and C. dubliniensis on conventional mycological media, could be distinguished from the other members of the C. albicans complex by appearance on chromogenic agar, and were incapable of forming chlamydospores. Here we present the detailed evaluation of epidemiological, phenotypic, and clinical features and antifungal susceptibility profiles of United Kingdom isolates of C. africana. Furthermore, we demonstrate that C. africana is significantly less pathogenic than C. albicans and C. dubliniensis in the Galleria mellonella insect systemic infection model.

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Section: Resultsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Epidemiology, Antifungal Susceptibility, and Pathogenicity of Candida africana Isolates from the United Kingdom

Borman¹,

Székely²,

Linton³

et al. 2013

J Clin Microbiol

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“…A variety of strain typing methods have been used for the differentiation of the C. parapsilosis family, including electrophoretic karyotype (EK) analysis (9,35), DNA fingerprinting with probe Cp3-13 (11), RAPD analysis (10,42), RFLP analysis (38,39), multilocus sequence typing (MLST) (36), and pyrosequencing (2). van Asbeck et al (38) compared three of these molecular typing methods, ITS sequencing, RAPD anal- ysis, and RFLP analysis, and indicated that ITS1 typing and RFLP analysis appear to have similar potentials to differentiate C. parapsilosis and both have discriminatory abilities superior to the discriminatory ability of RAPD analysis.…”

Section: Discussionmentioning

confidence: 99%

New Polymorphic Microsatellite Markers Able To Distinguish among Candida parapsilosis Sensu Stricto Isolates

et al. 2010

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Among the Candida species causing bloodstream infections, Candida parapsilosis is one of the most frequently isolated. The objective of the present work was the identification of new microsatellite loci able to distinguish among C. parapsilosis isolates. DNA sequences with trinucleotide repeats were selected from the C. parapsilosis genome database. PCR primer sets flanking the microsatellite repeats were designed and tested with 20 independent isolates. On the basis of the amplification efficiency, specificity, and observed polymorphism, four of the sequences were selected for strain typing. Two hundred thirty-three independent C. parapsilosis sensu stricto isolates were genotyped by using these markers. The polymorphic loci exhibited from 20 to 42 alleles and 39 to 92 genotypes. In a multiplex analysis, 192 genotypes were obtained and the combined discriminatory power of the four microsatellites was 0.99. Reproducibility was demonstrated by submission of subcultures of 4 isolates each, in triplicate, interspersed with unique numbers among a group of 30 isolates for blind testing. Comparison of the genotypes obtained by microsatellite analysis and those obtained by randomly amplified polymorphic DNA analysis, restriction fragment length polymorphism analysis, and internal transcribed sequence grouping was performed and showed that the microsatellite method could distinguish individual isolates; none of the other methods could do that. Related species, C. orthopsilosis and C. metapsilosis, were not confused with C. parapsilosis sensu stricto. These new microsatellites are a valuable tool for use for the differentiation of C. parapsilosis sensu stricto strains, vital in epidemiology to answer questions of strain relatedness and determine pathways of transmission.

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“…Molecular methods can be also applied for identification of isolates, e.g. pyrosequencing of target genes (Borman et al, 2009;. Further development of modified media and combinations of temperature, pH, aerobic/anaerobic conditions and moisture would probably increase the number of isolated yeasts, it is however laborious and very time consuming.…”

Section: Methods For Investigating Biodiversity Of the Yeasts From Gitmentioning

confidence: 99%

Biodiversity of Yeasts in the Gastrointestinal Ecosystem with Emphasis on Its Importance for the Host

Urubschurov¹,

Janczyk²

2011

The Dynamical Processes of Biodiversity - Case Studies of Evolution and Spatial Distribution

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Pyrosequencing Analysis of 20 Nucleotides of Internal Transcribed Spacer 2 Discriminates Candida parapsilosis , Candida metapsilosis , and Candida orthopsilosis

Cited by 42 publications

References 20 publications

Epidemiology, Antifungal Susceptibility, and Pathogenicity of Candida africana Isolates from the United Kingdom

Epidemiology, Antifungal Susceptibility, and Pathogenicity of Candida africana Isolates from the United Kingdom

New Polymorphic Microsatellite Markers Able To Distinguish among Candida parapsilosis Sensu Stricto Isolates

Biodiversity of Yeasts in the Gastrointestinal Ecosystem with Emphasis on Its Importance for the Host

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