FROM THE BENCH
Evolution of Mpox Testing in California and the Detection of the First Clade Ib Case in the Americas
By Jill K. Hacker, PhD, MPH, section chief, and William S. Probert, PhD, research scientist, Vaccine Preventable Diseases Section, Viral and Rickettsial Disease Laboratory, Center for Laboratory Sciences, California Department of Public Health
share the triplex MPXV rPCR protocol and reagents in support of other public health laboratories’ efforts to add or improve mpox testing capacity. This, in turn, alleviated much of the strain on VRDL staff, who had spent several weeks of long days to bring on MPXV-specific testing, while also running the LRN orthopoxvirus assays.
Colorized transmission electron micrograph of mpox particles( teal) found within an infected cell( brown), cultured in the laboratory. Image captured and color-enhanced at the NIAID Integrated Research Facility( IRF) in Fort Detrick, MD. Photo: NIAID.
The Viral and Rickettsial Disease Laboratory( VRDL) of the Center for Laboratory Sciences in the California Department of Public Health( CDPH) has provided testing for orthopoxviruses since 2002 using molecular tests provided through the US Centers for Disease Control and Prevention( CDC) Laboratory Response Network for Biological Threats( LRN-B) that were intended to help rule out smallpox. With early 2022 reports of global spread of clade IIb mpox cases and the subsequent identification of the first clade IIb case in the United States in May 2022, VRDL recognized the need to rapidly expand its testing toolbox to include monkeypox virus( MPXV)-specific molecular diagnostic tests and whole genome sequencing( WGS) capabilities. Within one week from detection of the first clade II case in California, VRDL began work to develop and validate a laboratory-developed triplex MPXV real-time PCR( rPCR) assay. This assay was based on CDC’ s generic and cladespecific assays and was designed to allow detection of MPXV and discrimination between clades I and II in a single well.
Celebrating Innovation …
An early hurdle was the lack of readily available poxvirus materials for contriving clade I specimens and assessing assay specificity. This was overcome by support from CDC’ s Poxvirus and Rabies Branch, which generously provided VRDL with control DNA and inactivated poxviruses. By mid-June 2022, VRDL had enough specimens from suspected mpox cases to support assay validation, and by mid-July 2022, validation of the triplex MPXV rPCR had been completed. With this new test, VRDL was able to confirm MPXV in non-variola orthopoxvirus( NVO)-positive specimens, thereby providing results in a timelier manner to local health departments and other clients, supporting epidemiological investigations and alleviating some of the testing burden from CDC’ s workload.
The truly collaborative nature of the 2022 public health laboratory mpox response cannot be overstated. As local health jurisdictions were overwhelmed with specimens for testing, some of which did not have access to the LRN-B orthopoxvirus assay, VRDL was able to
… Preparing for Failure …
An ongoing challenge to the implementation of molecular tools for MPXV detection has been the potential failure of MPXV rPCR tests that target non-essential genes, as does VRDL’ s triplex rPCR. Poxviruses are known to delete genes that are not essential for viral survival, a phenomenon associated with the emergence of viral variants. Indeed, early in the clade IIb outbreak, VRDL was informed by colleagues at the Los Angeles County Public Health Laboratory of a MPXV variant that had deleted the generic and clade II PCR target sequences( a PCR target“ dropout” mutant) and thus was not detectable with the available MPXV PCR assays that targeted these non-essential genes. While transient in spread, the emergence of this dropout mutant illustrated the risk in targeting non-essential sequences. VRDL dabbled with the idea of an assay that targeted genes within the central conserved region of the genome, but the transient nature of the clade II target dropout mutant and the lull in mpox samples coming to VRDL in 2023 put this on the back burner.
In January 2024, with the US Food and Drug Administration( FDA) laboratorydeveloped testing( LDT) rule looming, VRDL was approached by the LRN about collaborating to submit the triplex rPCR for an FDA Emergency Use Authorization. However, in February 2024, the clade Ib MPXV variant emerged, which was notable for deletion of the same commonly used clade I PCR target that
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