(P-TS-30) Detection And Serotyping Of Dengue Virus Infection Using Third Generation Sequencing Technology.

Dengue virus (DENV) is emerging or reemerging across the globe, posing a potential threat to blood supply, especially in endemic regions. As approximately 40–50% of DENV-infected individuals are asymptomatic, an infected individual could be accepted as a blood donor. Rapid, accurate and scalable diagnostics are key for viral detection but also for understanding the serotypes circulating during outbreaks and improving clinical outcomes. We utilized Oxford Nanopore technology (ONT) to investigate its performance detecting and serotyping DENV in specimens collected in a real epidemiological context. Our approach carries potential for application as a point-of-care assay in remote or low resource settings.

Study

Design/Methods:

24 plasma samples collected in Argentina during a 2016 DENV outbreak with a range of Ct values were tested. DENV RNA was quantified using the Altona RealStar RT qPCR kit (Altona Diagnostics, Germany). Complementary DNA (cDNA) was prepared using a Sequence Independent Single Primer Amplification (SISPA) approach (Ovation RNA-Seq System V2, Tecan Genomics, Switzerland) to increase assay sensitivity. Sequencing libraries were prepared using total amplified cDNA using ONT’s Ligation Sequencing Kit (SQK-LSK109) and Barcoding kit (EXP-PBC001). Samples were run on the ONT Mk1C, individually and in groups of 6 (multiplexed). Genome Detective software was used for alignment and to compute percentage reads mapped and genome coverage.

Results/Findings:

Single or multiplexed, all samples mapped to Dengue serotype 1 (DENV-1). Lower Ct values resulted in more viral reads and higher genome coverage. More reads correlated with increased genome coverage. Single runs proved most successful, 14/24 samples had over 91% genome coverage and except for one outlier, all samples had over 53% coverage. Multiplexed samples resulted in fewer reads and lower genome coverage, however these were sufficient to detect and determine DENV-1 in all samples. 12/24 samples had over 72% genome coverage and 7 of those had over 90% coverage. 4 samples had under 20% genome coverage.

Conclusions:

In this study, we reported the feasibility of using ONT technology to perform direct metagenomic sequencing without viral enrichment to identify the virus and specific serotype in DENV positive plasma samples. The assay showed high specificity, detecting DENV-1 in all samples. The portable ONT Mk1C device provided simplified real time sequencing and showed potential for use as a point-of-care assay.