Molecular screening for Candida orthopsilosis and Candida metapsilosis among Danish Candida parapsilosis group blood culture isolates: proposal of a new RFLP profile for differentiation.
Candida orthopsilosis and Candida metapsilosis are recently described species phenotypically indistinguishable from Candida parapsilosis . We evaluated phenotyping and molecular methods for the detection of these species among 79 unique blood culture isolates of the C. parapsilosis group obtained during the years 2004-2008. The isolates were screened by PCR amplification of the secondary alcohol dehydrogenase-encoding gene ( SADH) followed by digestion with the restriction enzyme Ban I, using C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139 and C. metapsilosis ATCC 96144 as controls. Isolates with RFLP patterns distinct from C. parapsilosis were characterized by sequence analysis of the ITS1-ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610 restriction 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. These results were confirmed by creating a phylogenetic tree based on concatenated sequences of SADH, ITS and 26S rRNA gene sequence regions. Optimal differentiation between C. parapsilosis, C. metapsilosis and 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 reference strains and 79 clinical isolates. In conclusion, reliable discrimination was obtained by PCR-RFLP profile analysis of the SADH gene after digestion with NlaIII but not with BanI. C. metapsilosis and C. orthopsilosis are involved in a small but significant number of invasive infections in Denmark.
Mirhendi
Hossein
H
Mycology and Parasitology Unit, Statens Serum Institut,Copenhagen, Denmark.|Department of Medical Mycology and Parasitology,Faculty of Public Health and National Institute of Health Research, TehranUniversity of Medical Sciences, Tehran, Iran.
Bruun
Brita
B
Department of Clinical Microbiology, HillerødHospital, Hillerød, Denmark.
Schønheyder
Henrik Carl
HC
Department of Clinical Microbiology, Aalborg Hospital,Aarhus University Hospital, Aalborg, Denmark.
Christensen
Jens Jørgen
JJ
Clinical Microbiology Unit, Statens Serum Institut,Copenhagen, Denmark.
Fuursted
Kurt
K
Department of Clinical Microbiology, Skejby Hospital,Aarhus University Hospital, Aarhus, Denmark.
Gahrn-Hansen
Bente
B
Department of Clinical Microbiology, Odense UniversityHospital, Odense, Denmark.
Johansen
Helle Krogh
HK
Department of Clinical Microbiology, Rigshospitalet,Copenhagen University Hospital, Copenhagen, Denmark.
Nielsen
Lene
L
Department of Clinical Microbiology, Herlev UniversityHospital, Herlev, Denmark.
Knudsen
Jenny Dahl
JD
Department of Clinical Microbiology, Hvidovre UniversityHospital, Hvidovre, Denmark.
Arendrup
Maiken Cavling
MC
Mycology and Parasitology Unit, Statens Serum Institut,Copenhagen, Denmark.
D000429:Alcohol Oxidoreductases / Q000235:genetics; D002175:Candida / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002177:Candidiasis / Q000175:diagnosis / Q000382:microbiology*; D016469:Fungemia / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D000429:Alcohol Oxidoreductases; C025192:isopropanol dehydrogenase (NADP)