Identification of clinically relevant fungi and prototheca species by rRNA gene sequencing and multilocus PCR coupled with electrospray ionization mass spectrometry.
BACKGROUND
Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.


METHODS
One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5' end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.


RESULTS
For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.


CONCLUSIONS
rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Xuan
X
Wang
Department of Medical Microbiology and Parasitology, Shanghai Medical College of Fudan University, Shanghai, China.
Yong-Feng
YF
Fu
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Rui-Ying
RY
Wang
Mycology Lab, Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China.
Li
L
Li
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Ya-Hui
YH
Cao
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Yan-Qiong
YQ
Chen
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Hua-Zhen
HZ
Zhao
Mycology Lab, Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China.
Qiang-Qiang
QQ
Zhang
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Ji-Qin
JQ
Wu
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Xin-Hua
XH
Weng
Department of Medical Microbiology and Parasitology, Shanghai Medical College of Fudan University, Shanghai, China.
Xun-Jia
XJ
Cheng
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Li-Ping
LP
Zhu
D005658:Fungi / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D005800:Genes, Fungal; D017343:Genes, Plant; D016133:Polymerase Chain Reaction; D011525:Prototheca / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D012335:RNA, Ribosomal / Q000235:genetics*; D017422:Sequence Analysis, DNA; D021241:Spectrometry, Mass, Electrospray Ionization
D012335:RNA, Ribosomal