Development and validation of a multiplexed quantitative PCR assay for clinical detection and surveillance of Oropouche virus
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Background
Oropouche virus (OROV) is an emerging vector-borne virus with rapidly expanding geographic range, increasing case counts, and growing evidence of severe outcomes including neuroinvasive disease and vertical transmission. Because OROV infection presents with nonspecific febrile illness that overlaps clinically with other viruses including dengue, zika, and chikungunya, accurate molecular diagnostics are essential for patient care and surveillance. Yet existing assays rely on single genomic targets and are vulnerable to detection failure as the virus evolves and reassorts.
Methodology/Principal Findings
To support diagnostic capacity, we developed and clinically validated a multiplexed qPCR assay targeting three regions of the OROV S segment, incorporating redundancy to preserve sensitivity across viral diversity while enabling robust clinical interpretation. The multiplex also includes an assay targeting RNaseP as an internal sample control to ensure adequate sample processing. We evaluated assay performance using both historical and contemporary OROV strains and validated the assay on contrived serum, plasma, and cerebrospinal fluid samples, assessing linearity, limit of detection (LOD), accuracy, specificity, precision, and sample stability. The assay met or exceeded all predefined acceptance criteria for clinical testing and achieved an LOD as low as 6 copies per reaction for contemporary outbreak strains. We further implemented a logic-based interpretation matrix that reduced false-positive risk while maintaining sensitivity near the analytical LOD.
Conclusions/Significance
Our assay sensitively and specifically detects OROV RNA in serum, plasma, and cerebrospinal fluid while incorporating safeguards against viral evolution and reassortment. The assay has been approved for use by CLIA at Nexus Medical Labs in 49 U.S. states, expanding access to timely OROV diagnostics in the United States and providing a durable framework for molecular detection of reassorting, rapidly evolving viruses as OROV continues to spread into new regions.
Author Summary
We present a fully validated multiplex qPCR assay for detection of Oropouche virus (OROV) in clinical samples. Our multiplex design includes three assays targeting distinct regions of the S segment, protecting against assay failure due to mutation dropout. In addition, we include a human internal control assay (RnaseP), which gives confidence to pathogen-negative clinical results by ensuring sample quality and adequate sample processing. The assay exhibits high sensitivity and specificity, and can be adopted by other labs should expanded OROV surveillance be needed. With geographic and case number expansion of OROV in recent years, this assay adds to the available tools for surveillance and clinical diagnosis, mitigating negative effects of the virus in the future.
