Rapid Differentiation of Vaccine and Wild-Type Measles Virus During an Ongoing Outbreak using a new molecular assay

Differentiating wild and vaccine measles virus (MeV) strains is critical for appropriate healthcare response during outbreak vaccination campaigns. Sequencing, the current standard, is expensive, time-consuming, and limited to specialized laboratories. We developed and implemented a novel differential MeV RT-qPCR assay to address these challenges, which we describe herein.

We designed a differential two-step assay; the first step, termed MRRV, distinguishes between rubella, wild MeV, and vaccine MeV. The second, termed "MeV differential", differentiates between genotypes A (vaccine), B and D.

The assay detects 10-50 copies per reaction and accurately classifies weak clinical samples (Cq > 32), with 100% accuracy, compared with the N gene sequencing gold standard. Evaluated on over 4,000 clinical samples retrospectively and during the 2025-2026MeV outbreak in Israel, the assay revealed that ∼9% of tested originated from vaccine strain. Among tested municipalities, those running active vaccination campaigns had significantly higher vaccine strain detections (>10 cases each) than those with routine vaccination (1-2 cases). Retrospective analysis of samples from 2018-2024 identified a mix of B and D strains. The novel assay successfully identified double infection cases (exposure to a wild strain shortly before or after vaccination).

The novel assay proved useful both as a diagnostic tool that enables focused, efficient healthcare response, and as an epidemiological tool that provides valuable information in real-time. This assay is faster, cheaper and more amendable for high throughput testing, compared with the gold-standard sequencing classification.