Lab Matters Summer 2025 | Page 14

FROM THE BENCH

to expand testing capacity across the US. Around the same time, SFPHL received primers and probes from the California Department of Public Health( CDPH) to implement a generic mpox assay. However, because the assay couldn’ t differentiate between Clade I and Clade II, specimens were considered select agents.

To address this, Masinde used CDC’ s published Clade I and II mpox sequences to develop a multiplex assay that could simultaneously target both clades.

Since obtaining an actual human sample of Clade I mpox wasn’ t possible, the laboratory prioritized creating a non-infectious specimen for validation. Masinde leveraged expertise gained while working in Silicon Valley biotech, where synthetic DNA is frequently used for assay validation. Synthetic DNA( gBlocks) consists of double-stranded fragments with a specific sequence targeted by the assay. These fragments also have known concentrations, which help determine the limit of detection for a test.

“ We had plenty of negative mpox specimens that we had previously tested,” Castro said.“ We just needed to make them positive for mpox Clade I.” She and Masinde designed synthetic DNA from the CDC-published sequences.

“ In situations where you don’ t have access to a unique specimen or pathogen, adaptation is key,” Masinde noted.“ The lesson here is that synthetic DNA is an invaluable tool for validating tests during emergencies. gBlocks are non-infectious, their concentration is known, and they can serve as positive controls.”

During the summer of 2022, the Los Angeles County Public Health Laboratory reported a cluster of mpox specimens with a 700bp deletion in the region targeted by the CDC’ s Clade II test. By then, SFPHL had already completed two validations: one for the generic mpox assay and another for the Clade I / II multiplex assay.

Because of the mutation, SFPHL had to send all negative specimens to CDPH for confirmation, as they were performing the non-variola orthopoxvirus test.

“ It was back to the drawing board,” said Masinde. SFPHL updated its multiplex Clade I / II assay to include the non-variola orthopoxvirus target, ensuring detection of all circulating strains.

Two years after the mpox Clade II outbreak started, a new outbreak of Clade Ib broke out in the Kivu province in the Democratic Republic of Congo. This time the Clade Ib variant had an 1100bp deletion in the region that was targeted by the CDC’ s Clade I test. SFPHL adapted and developed and validated a new multiplex assay that included targets for mpox Clade I, Clade Ib, non-variola orthopoxvirus and ribonuclease P( RNase P.)

Collaboration Leads to Validation

SFPHL is not part of the LRN-B, a network of laboratories responding quickly to emerging biological threats. However, with APHL’ s support, SFPHL consulted the US Food and Drug Administration( FDA) to ensure its test development approach complied with evolving laboratorydeveloped test( LDT) regulations.

The FDA confirmed that contriving specimens with synthetic DNA was acceptable for assessing test performance. Additionally, for specificity analysis, the FDA agreed that in silico evaluation using the National Center for Biotechnology Information( NCBI) database was sufficient to demonstrate no cross-reactivity with other viruses. These tools helped SFPHL validate its assay in less than two weeks.

“ This approach worked because the CDC allowed mpox testing in commercial laboratories with APHL’ s assistance,” Masinde said.“ This allowed our lab to multiplex the test— screening for mpox targets in one well and human controls in another. By multiplexing all targets, assay became more cost-effective, increased throughput, and reduced turnaround times.”

SFPHL also collaborated with APHL to share its approach to assay development and validation with other LRN-B laboratories.

“ Our assay was specific to San Francisco’ s instrumentation,” Masinde noted.“ And our SOP was tailored to our lab. But we worked with APHL to create a generic SOP to help other laboratories implement the assay using their available instruments,” Castro added.“ It’ s nice when another lab can say,‘ Okay, how did you do it?’ instead of reinventing the wheel.”

The Future of Testing

Public health threats like bird flu and measles continue to emerge, yet laboratories may lack essential materials to develop new assays. With this innovation, if a lab has access to the genetic sequence, it can validate and test samples efficiently.

“ We’ re looking to apply this technology to prepare for new and re-emerging diseases,” Castro said.“ For example, if we detected a positive measles specimen in San Francisco and needed to scale up testing, we could multiplex two primer sets for measles and create synthetic DNA to rapidly validate and implement a high-throughput assay.”

SFPHL’ s innovative test development approach underscores the vital role of laboratories in protecting public health. Without timely detection of infectious diseases like mpox, tracking and containing outbreaks is difficult. The staff’ s expertise, technological advances, and scientific ingenuity helped them respond swiftly to the 2022 mpox outbreak. Their development of synthetic mpox DNA provided a solution for obtaining positive specimens and offers a roadmap for future outbreak response. g