Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach

Nakamura, Shota; Yang, Cheng-Song; Sakon, Naomi; Ueda, Mayo; Tougan, Takahiro; Yamashita, Akifumi; Goto, Naohisa; Takahashi, Kazuo; Yasunaga, Teruo; Ikuta, Kazuyoshi; Mizutani, Tetsuya; Okamoto, Yoshiko; Tagami, Michihira; Morita, Ryoji; Maeda, Norihiro; Kawai, Jun; Hayashizaki, Yoshihide; Nagai, Yoshiyuki; Horii, Toshihiro; Iida, Tetsuya; Nakaya, Takaaki

Nucleic acid amplification tests (NATs) are increasingly being used for the diagnosis of viral infections. The most familiar formats use DNA or RNA target amplification methods, such as reverse transcription (RT) PCR, and have sensitivities that are greater than culture-or antigen-based procedures [3] . Loop-mediated isothermal amplification is more convenient and sensitive than PCR in amplifying DNA targets, and can be combined successfully with an RT step for RNA respiratory viruses. However, the wide variety of potential pathogens that elicit similar clinical symptoms and diseases makes the application of individual DNA-or RNA-based diagnostic assays both complex and expensive. Even multiplex PCRs are limited to 20-30 candidate pathogens, and may be confounded if viral evolution results in mutations at the primer binding sites [2] . DNA microarrays offer unprecedented opportunities for multiplexing; however, they are not widely implemented in clinical microbiology laboratories because of problems with sensitivity, throughput, and validation [2] . In addition, these microarrays are unavailable for unknown and/or unexpected microbes, as they require genetic information for each tested pathogen.