Molecular screening for Candida orthopsilosis and Candidametapsilosis among Danish Candida parapsilosis group blood cultureisolates: proposal of a new RFLP profile for differentiation
- 1 April 2010
- journal article
- Published by Microbiology Society in Journal of Medical Microbiology
- Vol. 59 (4) , 414-420
Abstract
Candida orthopsilosis and Candida metapsilosis are recentlydescribed species phenotypically indistinguishable from Candida parapsilosis. We evaluated phenotyping and molecular methods for the detection ofthese species among 79 unique blood culture isolates of the C. parapsilosis group obtained during the years 2004–2008. The isolates were screenedby PCR amplification of the secondary alcohol dehydrogenase-encoding gene (SADH) followed by digestion with the restriction enzyme BanI, using C. parapsilosis ATCC 22019, C. orthopsilosisATCC 96139 and C. metapsilosis ATCC 96144 as controls. Isolates withRFLP patterns distinct from C. parapsilosis were characterized bysequence analysis of the ITS1–ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610restriction enzymes were predicted in silico using 12 available sequences.By PCR-RFLP of the SADH gene alone, four isolates (5.1 %)had a pattern identical to the C. orthopsilosis reference strain.Sequence analysis of SADH and ITS (internal transcribed spacer)regions identified two of these isolates as C. metapsilosis. Theseresults were confirmed by creating a phylogenetic tree based on concatenatedsequences of SADH, ITS and 26S rRNA gene sequence regions. Optimaldifferentiation between C. parapsilosis, C. metapsilosisand C. orthopsilosis was predicted using digestion with NlaIII,producing discriminatory band sizes of: 131 and 505 bp; 74, 288 and 348 bp;and 131, 217 and 288 bp, respectively. This was confirmed using the referencestrains and 79 clinical isolates. In conclusion, reliable discrimination wasobtained by PCR-RFLP profile analysis of the SADH gene after digestionwith NlaIII but not with BanI. C. metapsilosisand C. orthopsilosis are involved in a small but significant numberof invasive infections in Denmark.Keywords
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