A-297 Feasibility of a novel Seegene Multiplex PCR panel for the simultaneous detection of HSV-1, HSV-2, VZV and Mpox: a focus on inclusion, confirmation and LOD assessment
Authors: Johnson T , Kim B , Yang D 
Published: October 2024

Abstract

Background
Characterized by atypical presentations of short prodromal symptoms, variations in clinical staging, non-uniform presentation of rash, and varied syndromes of proctitis and pharyngitis, the WHO declared the 2022 Mpox outbreak a global public health emergency in July 2022. The overlap of clinical presentation with other causes of vesicular lesions identified gaps in the diagnostic tests available for MPXV and highlighted the need for a multiplex PCR approach to the diagnosis of HSV, VZV, and MPXV among patients presenting with vesicular lesions. Currently, no FDA cleared multiplex approach offering a single unified approach to the diagnosis of HSV, VZV, and MPXV from a single sample exists. Multiplex assays offer a role in diagnostic support where clinicians are challenged to determine the causative agent because of overlap. Furthermore, no assay capable of providing MPXV clade II reporting, needed for compliance to select agent reporting rules, exist for routine clinical labs. This study outlines the initial feasibility of Seegene’s multiplex HSV, VZV, MPXV PCR assay, highlighting the LOD assessment from the 1st WHO International Standards, ZeptoMetrix® NATtrolTM Controls, and custom plasmids.

Methods
Gene targets representing conserved, differentiated regions of HSV-1, HSV-2, VZV, MPXV, and MPXV clade II were identified through phylogenetic analysis. A total of 28 oligonucleotide combinations, were applied to Seegene’s proprietary amplification and detection process creating a multiplex assay capable of 8 unique detection signals in 5 channels using the BioRad Opus® 96. 1st WHO International Standards (IS) for HSV-1/2, VZV, ZeptoMetrix® NATtrolTM and custom designed plasmids controls were titrated and spiked into Copan UTM supplemented with 5% HeLa cells and used to define target LOD panels ranging in concentrations tested with multiple replicates. Sample processing, DNA extraction, and PCR amplification were performed on the Seegene Starlet® automated liquid handler and BioRad® OPUS 96 using Seegene software.

Results
Detection rates for HSV-1 at target concentrations of 1x102 IU/ml (WHO) and 6.17x100 copies/ml (NATtrolTM) were 100%. Detection of HSV-1 with plasmid DNA at target concentrations of 1.25x101 was 95%. Detection rates for HSV-2 were 100% at 5x102 IU/ml (WHO) and 5.56x101 copies/ml (NATtrolTM). Detection of HSV-2 with plasmid DNA at target concentrations 2.5E1 was 100%. For VZV, detection rates of 100% were observed at LOD challenge pools comprising 1x102 IU/ml (WHO) and 1.85x101 copies/ml (NATtrolTM). Detection of VZV with plasmid DNA at target concentrations 2.5x101 was 95%. For MPXV, detection rates of 100% were observed when challenged with LOD pools at 1.67x101 copies/ml respectively. Detection of MPXV with plasmid DNA at target concentrations 6.25x100 was 95%. Confirmation of clade II reporting for MPXV was determined using phylogenetic analysis.

Conclusions
Standardize of Seegene’s multiplex HSV-1, HSV-2, VZV, MPXV assay using traceable quantitated standard resulted in &gt;95% detection rates of 6.17x100 copies/ml for HSV-1, 5.56x101 copies/ml for HSV-2, 1.85x101 copies/ml for VZV, and 1.67x101 copies/ml for MPXV respectively. A single multiplex PCR assay offers advantages for the simultaneous detection of HSV, VZV, and MPXV DNA in lesions specimens where clinical features present overlap and in patients whose clinical history is difficult to obtain.