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.
J Med Microbiol. 2010 Apr;59(Pt 4):414-420. doi: 10.1099/jmm.0.017293-0. Epub 2010 Jan 7.
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.
近平滑念珠菌和中间念珠菌是最近描述的种，表型上与拟滑念珠菌无法区分。我们评估了表型和分子方法，以检测 2004-2008 年间获得的 79 株独特的念珠菌属血培养分离株中的这些种。通过 PCR 扩增二级醇脱氢酶（SADH）基因，然后用限制酶 Ban I 进行消化，对分离株进行筛选，以念珠菌属 22019 为对照，近平滑念珠菌 96139 和中间念珠菌 96144。与拟滑念珠菌的 RFLP 模式不同的分离株通过 ITS1-ITS2、26S rRNA（D1/D2）和 SADH 区的序列分析进行鉴定。使用 12 个可用序列在计算机上预测了 3 种与 610 种限制酶中的每一种的限制模式。通过单独的 SADH 基因 PCR-RFLP，4 株（5.1%）的模式与近平滑念珠菌参考株相同。SADH 和 ITS（内部转录间隔区）区的序列分析将其中 2 株鉴定为中间念珠菌。这些结果通过基于 SADH、ITS 和 26S rRNA 基因序列区域的串联序列构建的系统发育树得到证实。使用 NlaIII 进行消化，预测最佳区分拟滑念珠菌、中间念珠菌和近平滑念珠菌，产生的鉴别带大小分别为：131 和 505 bp；74、288 和 348 bp；和 131、217 和 288 bp。使用参考株和 79 株临床分离株证实了这一点。总之，通过 NlaIII 消化后的 SADH 基因 PCR-RFLP 图谱分析可以获得可靠的区分，但 BanI 不行。中间念珠菌和近平滑念珠菌在丹麦涉及少数但重要的侵袭性感染。
