Sitterlé Emilie, Rodriguez Christophe, Mounier Roman, Calderaro Julien, Foulet Françoise, Develoux Michel, Pawlotsky Jean-Michel, Botterel Françoise
Département de Microbiologie, Créteil, Dynamyc, ENVA, UPEC Creteil, France.
Département de Microbiologie, Next-Generation Sequencing Platform pACT, IMRB CréteilFrance; Institut Mondor de Recherche Biomédicale U955Créteil, France.
Front Microbiol. 2017 Mar 7;8:334. doi: 10.3389/fmicb.2017.00334. eCollection 2017.
Some cases of fungal infection remained undiagnosed, especially when the pathogens are uncommon, require specific conditions for growth, or when several microbial species are present in the specimen. Ultra-Deep Sequencing (UDS) could be considered as a precise tool in the identification of involved pathogens in order to upgrade patient treatment. In this study, we report the implementation of UDS technology in medical laboratory during the follow-up of an atypical fungal infection case. Thanks to UDS technology, we document the first case of gastro-intestinal basidiobolomycosis (GIB) due to . The diagnosis was suspected after histopathological examination but conventional microbiological methods failed to supply proof. The final diagnosis was made by means of an original approach based on UDS. DNA was extracted from the embedded colon biopsy obtained after hemicolectomy, and a fragment encompassing the internal transcribed spacer (ITS) rDNA region was PCR-amplified. An Amplicon library was then prepared using Genome Sequencer Junior Titanium Kits (Roche/454 Life Sciences) and the library was pyrosequenced on a GS Junior (Roche/454 Life Sciences). Using this method, 2,247 sequences with more than 100 bases were generated and used for UDS analysis. represented 80% of the sequences, with an average homology of 98.8%. A phylogenetic tree with reference sequences confirmed the presence of (bootstrap value of 99%). : UDS-based diagnostic approaches are ready to integrate conventional diagnostic testing to improve documentation of infectious disease and the therapeutic management of patients.
一些真菌感染病例仍未得到诊断，尤其是当病原体不常见、需要特定生长条件或标本中存在多种微生物时。超深度测序（UDS）可被视为识别相关病原体的精确工具，以提升患者治疗水平。在本研究中，我们报告了在一例非典型真菌感染病例的随访过程中，UDS技术在医学实验室中的应用。借助UDS技术，我们记录了首例由……引起的胃肠道担子菌病（GIB）。组织病理学检查后怀疑有该诊断，但传统微生物学方法未能提供证据。最终诊断是通过基于UDS的原始方法做出的。从半结肠切除术后获得的包埋结肠活检组织中提取DNA，并对包含内转录间隔区（ITS）rDNA区域的片段进行PCR扩增。然后使用Genome Sequencer Junior Titanium试剂盒（罗氏/454生命科学公司）制备扩增子文库，并在GS Junior（罗氏/454生命科学公司）上对文库进行焦磷酸测序。使用这种方法，生成了2247条长度超过100个碱基的序列，并用于UDS分析。……占序列的80%，平均同源性为98.8%。与参考序列构建的系统发育树证实了……的存在（自展值为99%）。结论：基于UDS的诊断方法已准备好整合传统诊断测试，以改善传染病的记录和患者的治疗管理。
