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Develop highly sensitive monkeypox MDx assays using Specimen-specific TM Master Mixes
developing a highly sensitive and specific monkeypox assay. Although VTM is designed to maintain the stability of viruses, some VTMs which are formulated to inactivate viruses and bacteria contain components that inhibit the activity of nucleases 3 . These inhibitory components can impede a molecular assay if they are not removed or inactivated before testing. Meridian’s Lyo-Ready™ Direct DNA LAMP Saliva (MDX134) and Air-Dryable™ Direct DNA qPCR Saliva (MDX130) mixes are optimized to work on liquid samples containing a range of inhibitors, including VTM. An in-house study using primers and probes specifically designed for MPXV detection was conducted to demonstrate the mixes’ ability to amplify MPXV DNA in the presence of 10% VTM and 20% VTM. In addition, the sensitivity and reproducibility of the saliva mixes were compared against mixes from NEB and Themo to demonstrate their superior performance for MPXV detection. Specifically, two sets of primers were designed, one set to detect the monkeypox A-type inclusion (ATI) gene region (a unique 453-nucleotide residue in the West African strains but not in the Congo Basin strains) and a second set to detect the G2RG region within the tumor necrosis factor receptor gene (which is capable of detecting all known strains of MPXV). In the first study, the amplification sensitivity of Lyo-Ready™ Direct DNA LAMP Saliva was compared against NEB WarmStart ® and Thermo SuperScript™ IV in a titration assay spiked with 6,000, 600 or 60 copies of MPX DNA in the presence of 10% VTM or 20% VTM. Lyo-Ready™ Direct DNA LAMP Saliva exhibited the earliest time-to-results (TTR) across all samples, indicating greater sensitivity and reproducibility, particularly with low copies
include new specimen types and testing methods such as serology assays. Overall, early diagnosis, isolation, effective contact tracing, and vaccination strategies will be the key for effective control of this outbreak. Monkeypox Molecular Assays Molecular testing for monkeypox has generally been limited to governmental labs, due to the low prevalence of the disease in non-endemic countries. In the United States, there is only one 510K approved molecular test for Orthopoxviruses which is available from the Centre for Disease Control (CDC) 1 . A protocol recently released by the CDC for real-time PCR testing for monkeypox virus outlines the assay set-up and procedure recommended for monkeypox molecular screening 2 . The ideal specimen type is skin lesion material placed in viral transport media (VTM). In particular, the CDC and WHO recommend that at least two specimens from individuals with suspected monkeypox infection should be obtained from lesions, preferably from different locations on the body and from lesions with differing appearances. Oropharyngeal swabs may also be used, however data on the accuracy of this specimen type for diagnosis is limited for monkeypox. DNA extracted from whole blood is not considered a suitable specimen as the viraemic phase on the infection may have already passed at the time of rash onset. As testing begins to expand to commercial labs in order to increase the testing capacity, assay manufacturers are developing new molecular monkeypox assays that are based on the CDC and WHO recommendations. Selecting the best reagents that perform accurately with lesion swabs and VTM is a critical step in
Introduction Monkeypox virus (MPXV) is a zoonotic double-stranded DNA enveloped virus that belongs to the Orthopoxvirus genus of the Poxviridae family which also includes variola (VARV), cowpox (CPX), and vaccinia (VACV) viruses. The monkeypox virus is endemic to Africa with two distinct genetic groups (clades): the west African clade and the central African (Congo Basin) clade. In May 2022, a new monkeypox outbreak was reported by World Health Organization (WHO) and identified to be caused by a strain linked to the west African clade, which was also responsible for the 2018 – 2019 international outbreak. The new outbreak is spreading considerably more than previous outbreaks, with thousands of cases reported in over 60 countries, prompting the WHO to declare a global health emergency on July 23, 2022. Monkeypox has an incubation period of 4 to 21 days and the symptoms range from fever to respiratory distress with a characteristic rash and pustules/lesions developing a few days after the first symptoms. The virus is spread through contact with damaged skin, the respiratory tract or the mucous membrane (eyes, nose or mouth). Within the general population, the fatality rate of monkeypox can be up to 11% and is higher among young children, the elderly, pregnant women and immunocompromised individuals. The WHO currently recommends only testing individuals that meet the suspected case definition for monkeypox using nucleic acid amplification (NAAT) techniques such as qPCR or LAMP. As monkeypox continues to spread and more research is undertaken to understand the disease transmission, the WHO recommendations may change or evolve to
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Study 1: Speed and Reproducibility of Lyo-Ready™ Direct DNA LAMP Saliva (MDX134) with the ATI Gene and Viral Transport Media (VTM)
of the viral DNA. The second study examined the
reproducibility of Air-Dryable™ Direct DNA qPCR Saliva against Thermo TaqPath™ ProAmp Multiplex Master Mix in a serial dilution assay using 2,000, 200 or 20 copies of MPXV DNA (over 6 repeats for each dilution). The results illustrate the greater reproducibility of the Air-Dryable™ Saliva mix, leading to increased sensitivity and specificity of MPXV detection, particularly when only low copies of the viral DNA are present.
A)
A) Lyo-Ready™ Direct DNA LAMP Saliva (MDX134) was lyophilized with ATI primers and used for the amplification of a 10-fold serial dilution of MPXV DNA (6,000, 600 and 60 copies respectively) in the presence of 10% VTM and 20% VTM. The time to results (TTR) values were compared to the same primers and DNA with NEB WarmStart ® and Thermo SuperScript™ IV.
B)
10% VTM - 60 copies
20% VTM - 600 copies
Lyophilized MDX134 | NEB WarmStart ® | Thermo SuperScript™ IV RT-LAMP
B) In order to illustrate the results further, the results of Lyo-Ready™ Direct DNA LAMP Saliva (MDX134) (red), NEB WarmStart ® (orange) and Thermo SuperScript™ IV (black ) for the 60 copies of MPXV DNA in 10% VTM and 600 copies of MPXV DNA in 20% VTM are also displayed as amplification plots. The results illustrate the increased speed and specificity of the Lyo-Ready™ Direct DNA LAMP Saliva, with earlier TTR and greater reproducibility, particularly with low copies of the viral DNA.
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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
2000 copies 200 copies
2000 copies 200 copies
20 copies
20 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 monkeypox 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 monkeypox 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 monkeypox, 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 monkeypox testing remain unknown, as more information is uncovered on monkeypox transmission, it is likely that testing recommendations will evolve. Very recent research suggests that monkeypox infections can be asymptomatic, which indicates the potential need for wider
testing and isolation policies among people exposed to the virus 4 . If screening expands to individuals exposed to active monkeypox 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 monkeypox outbreaks, such as the one in 2003 in the United States, serologic assays provided significant diagnostic support. Monkeypox 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 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. Aden T.A., Blevins P., York S.W., et al. Rapid Diagnostic Testing for Response to the Monkeypox Outbreak — Laboratory Response Network, United States, May 17–June 30, 2022. MMWR Morb Mortal Wkly Rep 2022;71:904-907. http://dx.doi.org/10.15585/ mmwr.mm7128e1. 2. Centers for Disease Control & Prevention Poxvirus & Rabies Branch (PRB), Test Procedure: Monkeypox virus Generic Real-Time PCR Test (https://www.cdc.gov/poxvirus/ monkeypox/pdf/pcr-diagnostic-protocol-508.pdf) 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, Van Dijck, C., Kenyon, C. et al. Asymptomatic monkeypox 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., Reynolds, M., Braden, Z. et al. Characterization of Acute-Phase Humoral Immunity to Monkeypox: Use of Immunoglobulin M Enzyme-Linked Immunosorbent Assay for Detection of Monkeypox Infection during the 2003 North American Outbreak. Clinical and Vaccine
monkeypox
Immunology. 2005; 12: 7. https://doi. org/10.1128/CDLI.12.7.867-872.2005
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