A targeted sequencing assay for West Africa specific infectious diseases

Rapid and precise diagnostic testing is required for efficacious treatment and patient isolation. Pathogen-specific ELISAs and real-time PCR are commonly used as a first-line diagnostic due to accuracy, cost, and ease of use.... [ view full abstract ]

Rapid and precise diagnostic testing is required for efficacious treatment and patient isolation. Pathogen-specific ELISAs and real-time PCR are commonly used as a first-line diagnostic due to accuracy, cost, and ease of use. Increasingly, next-generation sequencing (NGS) is being used for pathogen detection in cases where initial testing (ex. by real-time PCR) is negative. Agnostic sequencing, however, is challenging for diagnostics due to the amount of host nucleic acid in the sample and a lack of specificity generally needed for FDA approval. To address these limitations of agnostic NGS for infectious disease diagnostics, we designed, optimized, and characterized a multiplexed, amplicon-based NGS assay targeting 14 pathogens found in West Africa that can have a similar clinical presentation (acute febrile illness). Utilizing a primer multiplex of 18 primers for target amplification and sequencing on the MiSeq, we detected all 14 pathogens on the panel including Chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus serotypes 1, 2, 3, and 4, Ebola virus, Lassa virus, Marburg virus, Plasmodium falciparum, Rift Valley fever virus, West Nile virus, yellow fever virus, and Zika virus. Full assay characterization identified the limit of detection in matrix (serum) for each pathogen, inclusivity and exclusivity testing, and co-infection testing. Based on these data, we developed and characterized a sensitive, multiplexed NGS assay that can be used to detect multiple pathogens found in West Africa.