Evaluation of two point-of-care molecular diagnostic platforms for rapid detection of equine Hendra virus

Abstract

Hendra virus (HeV) is a lethal zoonotic pathogen endemic to eastern Australia, posing significant risks to equine and human health. Rapid field detection of HeV enables timely intervention and outbreak management. This study evaluated candidate point-of-care (POC) molecular diagnostic platforms for HeV detection in equine samples: including a loop-mediated isothermal amplification (DARQ RT-LAMP) assay and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Comparative analytical evaluation demonstrated that RT-qPCR exhibited superior analytical sensitivity relative to DARQ RT-LAMP, with a limit of detection of 1 copy/µL compared with 1,000 copies/µL, respectively. On this basis, DARQ RT-LAMP was not progressed beyond initial analytical evaluation due to insufficient sensitivity for the intended application. Bayesian Latent Class Model (BLCM) analysis estimated diagnostic sensitivity of 63.1% (95%PI 48.8–76.1%) and 80.4% (95%PI 67.6–90.2%) for RT-qPCR with HUDSON-prepared samples and extracted RNA, respectively, with identical specificity of 96.5% (95%PI 85.9–99.9%) for both sample types in virus transport medium. In 10% EDTA blood, diagnostic sensitivity was 71.3% (95%PI 54.2–85.1%) and 88.3% (95%PI 74.2–97.3%), respectively, with comparable specificity. Preliminary assessment of repeatability and reproducibility was promising (CVs <10%), although further field studies are required. These findings demonstrate that RT-qPCR, combined with rapid HUDSON sample preparation, provides a feasible molecular POC approach for preliminary rule-in or exclusion of HeV infection in horses while confirmatory laboratory testing is pending, supporting early risk management and reduced occupational exposure.