Meridian Monkeypox App Note 0824

Study 2: Sensitivity and Reproducibility of Air-Dryable™ Direct DNA qPCR Saliva (MDX130) with the ATI and G2R-G Gene

ATI gene

G2R-G

Human RNaseP

2000 copies 200 copies

20 copies

Air-Dryable™ Direct DNA qPCR Saliva (MDX130) ( red ) was dried with ATI primers, G2R-G or human RNaseP (as an internal control) and used for the amplification of a 10-fold serial dilution of MPXV DNA (2,000, 200 and 20 copies respectively), using 6 repeats of each dilution. The results were compared to the same primers and DNA for Thermo TaqPath™ ProAmp Multiplex Master Mix ( black ). The results illustrate the increased sensitivity and specificity of the Air-Dryable™ Direct DNA qPCR Saliva, with better sensitivity, specificity and reproducibility, particularly with low copies of the viral DNA.

Conclusion As the mpox outbreak continues to grow, the need for highly sensitive and specific detection assays increases. We have shown that both our qPCR and LAMP Sample- specific™ Saliva mixes can be used to develop fast, sensitive and reproducible assays for the detection of mpox using specimens containing VTM. These mixes are fully optimized and only require the addition of primers and probes as demonstrated in our studies. Ready-to-use mixes such as ours, enable assay manufacturers to fast-track their assay development for mpox, avoiding the need for extensive optimization of reagents compatible with inhibitors present in VTM solutions. In addition, the mixes are both formulated to be dried down, either by lyophilization (Lyo-Ready) or air-dried (Air-Drayble™), simplifying the process of creating ambient- temperature stable assays. Assays that can be shipped and stored at room-temperature should be particularly beneficial in remote regions such as Africa, where testing facilities and other resources are limited but the virus is endemic. Although the future recommendations for mpox testing remain unknown, as more recommendations will evolve. Very recent research suggests that mpox infections can be asymptomatic, which indicates the potential need for wider testing and information is uncovered on mpox transmission, it is likely that testing

isolation policies among people exposed to the virus 4 . If screening expands to individuals exposed to active mpox cases, then testing may need to include molecular screening using blood specimens and serology-based immunoassays that can detect acute subclinical and asymptomatic infections. In previous mpox outbreaks, such as the one in 2003 in the United States, serologic assays provided significant diagnostic support. Mpox IgM capture assays were able to distinguish between vaccinated and unvaccinated individuals with a sensitivity and specificity of 95% 5 . Overall, a coordinated, worldwide effort will be required to stop the mpox outbreak, and a significant scale-up in testing will be most likely be required in regions around the globe, especially where case numbers are the highest.

1. FDA “Monkeypox (mpox) and Medical Devices”. Retrieved from: https://www.fda.gov/ medical-devices/emergency-situations-medical- devices/monkeypox-mpox-and-medical-devices 2. World Health Organization. Diagnostic Testing for the Monkeypox Virus (MPXV): Interim Guidance. 10 May 2024. Retrieved from: https:// iris.who.int/bitstream/handle/10665/376952/ WHO-MPX-Laboratory-2024.1-eng. pdf?sequence=1 3. Daum L.T., et al. A clinical specimen collection and transport medium for molecular diagnostic and genomic applications. Epidemiol. Infect. 2011; 139: 1764e73. https://doi.org/10.1017/ S0950268810002384 4. Baetselier, I.D, et al . Asymptomatic mpox virus infections among male sexual health clinic attendees in Belgium. medRxiv 2022.07.04.22277226. https://doi. org/10.1101/2022.07.04.22277226 5. Karem, K.L., et al . Characterization of Acute-Phase Humoral Immunity to Mpox: Use of Immunoglobulin M Enzyme-Linked Immunosorbent Assay for Detection of Mpox Infection during the 2003 North American Outbreak. Clinical and Vaccine Immunology. 2005; 12: 7. https://doi.org/10.1128/ CDLI.12.7.867-872.2005

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