Lab Matters Fall 2025 | Page 13

FROM THE BENCH

VRDL was fortunate to be included in an APHL MPXV WGS Working Group. In parallel with triplex rPCR assay validation efforts, VRDL investigated a metagenomic approach to MPXV WGS. While successful for samples with high viral loads, the method lacked analytical sensitivity and sample throughput capacity. Through participation in the APHL MPXV Working Group, VRDL learned about a tiled amplicon sequencing approach developed by the Grubaugh laboratory at Yale University and implemented this procedure in late July 2022. With this tool, the laboratory was able to track MPXV lineages and the emergence of new variants and, in partnership with other state and local public health laboratories and CDC, investigate the influence of VP37 mutations on resistance to the antiviral compound, Tecovirimat. VRDL’ s work on this initiative has also recently been published in Frontiers.

VRDL had chosen for its assay, also located within the variable terminal region of the MPXV genome. This clade Ib variant was the cause of a growing outbreak in Africa and raised concern over the ability to detect this more severe clade of mpox. While the generic MPXV target in VRDL’ s triplex assay would detect clade Ib, the clade I target( now considered clade Ia) would not. Mid-2024, an Mpox Assay Development Working Group was convened by APHL to provide a forum for interested public health laboratories to discuss clade I assay development strategies.

To minimize the risk of PCR target dropout and enable identification of both clade Ia and Ib strains, VRDL reimagined its MPXV assay to target essential genes( i. e., genes required for poxvirus propagation) located within the central conserved region of the genome. The new assay was designed as a multiplex rPCR capable of detecting four analytes: orthopoxviruses( panOPX), MPXV, clade I MPXV and clade II MPXV. While conceptually appealing, the identification of clade-specific sequences within these highly conserved regions proved to be extremely challenging, necessitating the use of a single nucleotide polymorphism( SNP) assay for the clade I target. Importantly, this SNP is conserved among both clade Ia and Ib strains. Validation of the mpox essential genes quadplex( MpoxEG4-plex) rPCR assay commenced in late October 2024, again with substantial reagent support from the CDC Poxvirus and Rabies Branch.

… and Transforming for Future Success

In late November 2024, VRDL was notified of a suspect clade I mpox case by the San Mateo County Health Department based on NVO-positive, clade II-negative PCR results obtained at a commercial laboratory, clinical signs and symptoms, and recent travel to East Africa. San Mateo County Health professionals collected additional specimens that same day and transported them to VRDL for testing. The VRDL team quickly applied tests to determine if this was indeed clade I. Within hours, specimens were concurrently confirmed to be NVO / OPX-positive using LRN assays; negative for the generic, clade Ia and clade II targets with the triplex MPXV rPCR assay; and positive for the panOPX, MPXV and clade I analytes with the MpoxEG4-plex rPCR assay. Following WGS confirmation by both VRDL and CDC, the case was deemed the first clade Ib case reported in the Americas. Investigation of 83 contacts by the CDPH clade I mpox response team and the San Mateo County Health Department identified no secondary cases, with testing provided by Santa Clara County and San Mateo County Public Health Laboratories and VRDL. VRDL completed validation of the laboratory-developed MpoxEG4-plex rPCR assay for diagnostic use in January 2025.

Describing this mpox journey gives VRDL the opportunity to highlight the collaborative nature of public health laboratory work— whether it is the can-do attitude of the amazing laboratory team, the generous contributions of hard-working CDC colleagues, or the“ yes we can, tell us what you need” efforts of California’ s local public health laboratories and APHL. Throughout, VRDL was bolstered by colleagues at the CDC Poxvirus and Rabies Branch, and the public health laboratories of San Francisco, Santa Clara and Los Angeles counties, among others. The future of testing in public health is strongly rooted in molecular assays. Without the collective drive to advance efforts to detect emerging pathogens, this story would not be as successful. The ease of sharing knowledge, reagents and protocols between laboratories strengthens crucial scientific ties, especially as expertise in assay development and technical skills continue to be key to providing a way to stay at the forefront of pathogen diagnostics in an ever-changing microbial world. g