# Development of a dual-target measles virus PCR assay and testing trends at a national reference laboratory

## TL;DR

A new PCR test for measles virus distinguishes between wild-type and vaccine strains, aiding diagnosis and tracking during a U.S. outbreak.

## Contribution

Development and validation of a dual-target PCR assay for differentiating wild-type and vaccine strains of measles virus.

## Key findings

- Measles virus was detected in 16 specimens, with 6 wild-type and 10 vaccine strains identified.
- Children under 10 years old were most frequently tested and had the majority of vaccine strain detections.
- PCR showed low positive agreement (25%) but high negative agreement (98%) with IgM serology when paired testing was available.

## Abstract

In 2025, measles cases in the United States have reached their highest level since the disease was officially declared eliminated in 2000. Molecular assays may assist in the early diagnosis of measles and improve contact tracing efforts. In this manuscript, we describe the validation of a real-time PCR assay on the Hologic Panther Fusion Open Access system for the detection of measles virus (MeV) and differentiation between wild-type and vaccine strains. After implementation, we examined 3 months of clinical testing data to understand testing trends and utilization. Over the study period, a total of 525 tests from 491 patients were performed. MeV was detected in 16 specimens with 6 wild-type and 10 vaccine strain identifications. Children less than 10 years old constituted the largest proportion of tested individuals (54.3%) and vaccine strain detections (9/10, median age 1.2 years), while wild-type infections were observed in individuals aged 20–50 (6/6, median age 32.6 years). Those with vaccine strain detected had significantly higher Ct values for the pan-measles target versus wild-type infections (33.6 vs 28.3; P-value < 0.05). Only 4.2% of patients in our cohort received paired serologic and molecular measles testing. When paired data were available, PCR had a positive agreement of 25% and a negative agreement of 98% with IgM results. Molecular testing for MeV with the ability to differentiate wild-type strains from vaccine strains is a helpful tool in the response to measles re-emergence.

The 2025 U.S. measles outbreak comes at a challenging time for public health in America. As vaccine hesitancy increases and resources are withdrawn from national and state public health laboratories, historically low incidence diseases develop into nationwide outbreaks that require increased testing capacity. In response to this need, our national reference laboratory developed a measles PCR assay that allows for the detection and separation of vaccine from wild-type strains. The assay was launched on the Hologic Panther fusion system to improve throughput and reduce turnaround times. In this research article, we describe the design of our assay, validation results, and early clinical performance.