B-231 Development and Evaluation of a Lyophilized Mpox Virus qPCR Assay

Abstract

Abstract Background Increased cases of monkeypox virus (MPXV) have been reported to WHO from 110 countries. As of January 16, 2023, a total of 84 733 laboratory confirmed and 1354 probable cases, including 80 deaths, have been reported to WHO. The US is assessed as high-risk region. Real-time PCR, with its sensitivity and specificity, is the preferred lab test technique. Since gene mutation has been observed in MPXV, developing assays with more than one target was recommended by the FDA. Methods A lyophilized qPCR multiplex assay was developed for MPXV detection. This assay contains primers and (1) MGB probe targeting highly conserved MPXV sequence (GeneBank AF380138 F3L), (2) BHQ probe targeting non-variola orthopoxvirus (OPX) sequence, and (3) a probe targeting a selected human house-keeping gene as an internal control. Furthermore, external positive and negative controls were included. A MPXV positive clinical sample from human skin lesions was used for the assay validation. The viral copy number of the sample was determined as 1×107 copies/mL using the standard curve method. Two hundred µL of samples were processed in the KingFisherTM Flex system. The PCR reaction consisted of 5 µL of DNA sample and 20 µL reaction mix and was run on 7500 Fast Dx Real-Time PCR System (Life Technologies). The limit of detection (LoD) was determined as the concentration at which at least 95% of samples were tested positive. A total of 230 contrived samples with concentrations lower than LoD were used to determine cut-off value. Inclusivity and exclusivity studies were conducted by in silico analysis of 80 sequences of MPXV (clade I), 3026 of MPXV (clade II), 10 of camelpox virus, 93 of cowpox virus, 6 of ectromelia virus, and 116 of vaccinia virus (NCBI and GISAID accessed on 10/03/22). Clinical evaluation of the assay was conducted using 80 contrived clinical samples in unique negative skin lesion matrix, including 40 positives (five samples at 5× LOD, five at 2.5×, ten at 1.5×, and twenty at 1×), along with 40 negatives. The positive (PPA) and negative percent agreement (NPA) of the assay were calculated. The study was approved by BRANY IRB. Results The qPCR assay had a cut-off Ct value of 40, and a LoD of 500 copies/mL for both MPXV and OPX with good inclusivity and exclusivity. Clinical evaluation for MPXV yielded a PPA and NPA of 94.9% (95% CI: 83.1%–98.6%) and 100% (89.9%–100%), respectively. For OPX, the PPA and NPA were 92.3% (79.7%–97.4%) and 100% (89.9%–100%), respectively. Conclusions Both analytical and clinical performances of the developed MPXV qPCR assay were good. Lyophilized reagents contain all required components, providing longer shelf-life and easier transportation. MPXV is the only member of the OPX genus known to be circulating among humans in the US currently. This assay as designed could potentially cross-detect different non-variola OPX, such as vaccinia virus and cowpox virus. Healthcare providers should always contact the local public health authorities for more guidance. Furthermore, this study was limited to lesion swab specimens and performance evaluation with additional specimen types is warranted.