A novel multiplexed assay to diagnose Mpox and other viruses

By Neha MathurReviewed by Benedette Cuffari, M.Sc.Feb 6 2023

In a recent article published in the Journal of Clinical Virology Plus, researchers evaluate the performance of a research use only (RUO) assay to identify Mpox viral infection.

Study: Mpox infection investigation using multiplexed syndromic diagnostics: Evaluation of an AusDiagnostics multiplexed tandem PCR (MT-PCR) syndromic panel. Image Credit: PolakPhoto / Shutterstock.com

Diagnosing Mpox

After reaching the peak number of cases in the United Kingdom in July 2022, the prevalence of Mpox cases continues to decline. However, the 2022 Mpox outbreak outside of endemic areas showed that this virus likely causes a viral rash illness similar to other viruses.

The recent outbreak demonstrated that nucleic acid amplification tests (NAAT) are insufficient in detecting the genome of Mpox in patient samples, thus suggesting that the Mpox virus should now be incorporated into multiplexed syndromic panels.

In the current study, researchers evaluate the performance of an ROU syndromic panel named AusDiagnostics Multiplexed Tandem Polymerase Chain Reaction (MT-PCR). MT-PCR detects a Mpox virus target in addition to a panel of several pathogens that also cause vesicular rash and infect the central nervous system (CNS).

The researchers compared the utility of MT-PCR against quantitative real-time PCR (qPCR) to identify the presence of pathogens other than Mpox early in the outbreak and contemporary sample panels.

About the study

Two sets of vesicle swab samples were obtained from patients who required Mpox virus genome detection for routine clinical care. Panel 1 samples comprised residual nucleic acids submitted for Mpox virus detection at the Rare and Imported Pathogens Laboratory (RIPL) in the U.K. between May 15, 2022, and June 24, 2022, which was early in the recent Mpox outbreak.

Panel 2 samples, which were otherwise referred to as contemporary samples, comprised residual nucleic acids submitted for Mpox virus detection by qPCR laboratory-developed test (MPOX LDT) between August 30, 2022, and September 5, 2022.

MT-PCR was performed on the samples as part of a comprehensive multiplexed panel with 15 pathogen targets, some of which included human herpesvirus 1, 2, and 6. The assay targeted the F3L gene of the Mpox virus genome and required 10 µL of nucleic acid material and a single set of reagents. Both positive and negative percent agreement (PPA and NPA, respectively) were calculated, in addition to determining the overall accuracy of the AusDiagnostics MT-PCR Mpox assay.

Study findings

Out of the 175 samples tested, the MT-PCR assay detected the Mpox virus genome in 21.9% of panel 2 and 51.1% of panel 1 samples. Several other pathogens causing vesicular rash syndromes in Mpox cases were also co-detected.

The AusDiagnostics RUO MT-PCR panel was associated with an accuracy of 98.9%, a sensitivity of 94.2%, and 100% specificity. Its performance was comparable to an LDT qPCR assay used to detect the Mpox virus in nucleic acids extracted from swab specimens. Accordingly, the assay had a limit of detection (LOD) of 35 copies of the Mpox genome in residual nucleic acid specimens.

The MT-PCR Mpox assay yielded cycle threshold (C_T) values within the range of 9.58 and 35.07, with an average of 17.2. These values are slightly lower but close to the results obtained through a Mpox LDT qPCR.

Apart from the Mpox virus, the MT-PCR assay enabled samples to be screened for five additional pathogens that routinely cause a vesicular rash. In 18 and 29 samples of panels 1 and 2, respectively, which otherwise amounted to 26.8% of total samples, the assay detected at least one such pathogen.

Conclusions

The laboratory where the team collected test samples was a geographic location that was excessively affected during the Mpox outbreak in the U.K. Thus, the values reported in the current study do not reflect Mpox viral prevalence observed elsewhere.

Clinical laboratories did not perform diagnostic testing on 13.7% of samples infected with a pathogen attributable to a vesicular rash. The MT-PCR assay co-detected the Mpox virus with other vesicular rash pathogens in 12.8% of samples, with the enterovirus being the most frequently detected in 5.7% of samples.

Amid the Mpox outbreak, other viral pathogens causing a vesicular rash syndrome also remained prevalent in several patients, which highlights the advantages associated with using multiplexed panels under evaluation. Thus, Mpox diagnostics could be easily embedded within multiplexed syndromic panels and used for managing infections in areas where the Mpox virus is endemic. Furthermore, clinical virology laboratories might benefit from multiplexed syndromic panels that alleviate the need for multiple single pathogen assays.