Arbefeville S, Harris A, Ferrieri P
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, MMC 609 Mayo, 420 Delaware St. S.E., Minneapolis, MN 55455, USA.
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, MMC 609 Mayo, 420 Delaware St. S.E., Minneapolis, MN 55455, USA.
J Microbiol Methods. 2017 Sep;140:40-46. doi: 10.1016/j.mimet.2017.06.015. Epub 2017 Jun 21.
Fungal infections cause considerable morbidity and mortality in immunocompromised patients. Rapid and accurate identification of fungi is essential to guide accurately targeted antifungal therapy. With the advent of molecular methods, clinical laboratories can use new technologies to supplement traditional phenotypic identification of fungi.
The aims of the study were to evaluate the sole commercially available MicroSEQ® D2 LSU rDNA Fungal Identification Kit compared to the in-house developed internal transcribed spacer (ITS) regions assay in identifying moulds, using two well-known online public databases to analyze sequenced data.
85 common and uncommon clinically relevant fungi isolated from clinical specimens were sequenced for the D2 region of the large subunit (LSU) of ribosomal RNA (rRNA) gene with the MicroSEQ® Kit and the ITS regions with the in house developed assay. The generated sequenced data were analyzed with the online GenBank and MycoBank public databases.
The D2 region of the LSU rRNA gene identified 89.4% or 92.9% of the 85 isolates to the genus level and the full ITS region (f-ITS) 96.5% or 100%, using GenBank or MycoBank, respectively, when compared to the consensus ID. When comparing species-level designations to the consensus ID, D2 region of the LSU rRNA gene aligned with 44.7% (38/85) or 52.9% (45/85) of these isolates in GenBank or MycoBank, respectively. By comparison, f-ITS possessed greater specificity, followed by ITS1, then ITS2 regions using GenBank or MycoBank. Using GenBank or MycoBank, D2 region of the LSU rRNA gene outperformed phenotypic based ID at the genus level. Comparing rates of ID between D2 region of the LSU rRNA gene and the ITS regions in GenBank or MycoBank at the species level against the consensus ID, f-ITS and ITS2 exceeded performance of the D2 region of the LSU rRNA gene, but ITS1 had similar performance to the D2 region of the LSU rRNA gene using MycoBank.
Our results indicated that the MicroSEQ® D2 LSU rDNA Fungal Identification Kit was equivalent to the in-house developed ITS regions assay to identify fungi at the genus level. The MycoBank database gave a better curated database and thus allowed a better genus and species identification for both D2 region of the LSU rRNA gene and ITS regions.
真菌感染在免疫功能低下患者中会导致相当高的发病率和死亡率。快速准确地鉴定真菌对于指导精准靶向抗真菌治疗至关重要。随着分子方法的出现，临床实验室可以使用新技术来补充传统的真菌表型鉴定方法。
本研究的目的是评估唯一可商购的MicroSEQ® D2 LSU rDNA真菌鉴定试剂盒与内部开发的内转录间隔区（ITS）区域检测方法在鉴定霉菌方面的效果，并使用两个知名的在线公共数据库分析测序数据。
从临床标本中分离出85种常见和不常见的临床相关真菌，使用MicroSEQ®试剂盒对核糖体RNA（rRNA）基因大亚基（LSU）的D2区域进行测序，使用内部开发的检测方法对ITS区域进行测序。生成的测序数据使用在线GenBank和MycoBank公共数据库进行分析。
与一致鉴定结果相比，使用GenBank或MycoBank时，LSU rRNA基因的D2区域将85株分离株中的89.4%或92.9%鉴定到属水平，完整ITS区域（f-ITS）分别鉴定到96.5%或100%。在将种水平的鉴定结果与一致鉴定结果进行比较时，LSU rRNA基因的D2区域在GenBank或MycoBank中分别与这些分离株中的44.7%（38/85）或52.9%（45/85）匹配。相比之下，使用GenBank或MycoBank时，f-ITS具有更高的特异性，其次是ITS1，然后是ITS2区域。在属水平上，使用GenBank或MycoBank时，LSU rRNA基因的D2区域优于基于表型的鉴定方法。在种水平上，将LSU rRNA基因的D2区域和ITS区域在GenBank或MycoBank中的鉴定率与一致鉴定结果进行比较，f-ITS和ITS2的表现超过了LSU rRNA基因的D2区域，但使用MycoBank时，ITS1与LSU rRNA基因的D2区域表现相似。
我们的结果表明，MicroSEQ® D2 LSU rDNA真菌鉴定试剂盒在属水平上鉴定真菌的效果与内部开发的ITS区域检测方法相当。MycoBank数据库提供了一个更好的整理数据库，因此对于LSU rRNA基因的D2区域和ITS区域都能实现更好的属和种的鉴定。
