Everything you need to know about testing monkeypox

  • Published: 4 July 2022
There is a viral disease going around this year called monkeypox. It is endemic to West Africa from where it spread to other regions of the world that are currently non-endemic [1]. While monkeypox was first discovered already in 1958 and first identified in humans in 1970, it hadn’t widely spread outside of Africa until now [2]. To prevent this virus from spreading any further it is crucial to implement reliable diagnostic and testing methods.

Monkeypox virus (MPXV) belongs to the Orthopoxvirus genus in the family Poxviridae [2]. Currently there is a variation of different PCR kits available for detecting monkeypox (MPXV) or orthopoxvirus (OPXV) [1]. Most of them are for research use only and none is independently validated [1]. This shows that there is much room for improvement, especially since symptomatic diagnosis in case of monkeypox is complicated [1].

Testing according to the WHO guidelines

Getting the specimen

The recommended specimen type for laboratory confirmation of monkeypox is skin lesion material, including swabs of lesion surface and/or exudate, roofs from more than one lesion, or lesion crusts. Both dry swabs and swabs placed in viral transport media (VTM) can be used. Two lesions of the same type should be collected in one single tube, preferably from different locations on the body and which differ in appearance. Lesions, crusts and vesicular fluids should not be mixed in the same tube. In addition to a lesion specimen, the collection of an oropharyngeal swab is encouraged. However, data on the accuracy of this specimen type for diagnosis is limited for monkeypox, therefore a negative throat swab specimen should be interpreted with caution. [3]

Reagents

Reagents should be stored according to manufacturer recommendations. There are a number of primer and probe sequence sets for PCR assays for OPXV and specifically MPXV that have been published in the literature and can be used for in house development of assays in laboratories with appropriate capacities [4][5][6]. PCR kits detecting OPXV or specifically MPXV are under development [7][8], but no commercial PCR or serology kits are currently widely available. Positive control material for PCR assays can be ordered from specialized initiatives [9]. 

Best practice for the use of reagents:
  1. Positive control should be included at a low (above the limit of detection), but easily detectable concentration. 
  2. Inclusion of quality control materials where possible can assist in controlling for any assay issues. 
  3. Controls should provide information about 
    1. sample quality
    2. nucleic acid quality
    3. process quality. 
  4. PCR can be extremely sensitive so efforts should be made to limit contamination.
  5. Negative controls on every run should be utilized to ensure contamination has not occurred.
  6. Sample integrity controls (e.g. RNase P), extraction, positive and inhibition controls can help in distinguishing a false negative from a true negative.
  7. Controls should be utilized following laboratory SOPs. If any of the assay controls fail, testing should be repeated. [3]

Solis BioDyne recommendations for the best results

5x concentration of the reagent is beneficial, if you would like to add a bigger sample size in the reaction.

Advantages of 5x-concentrated mixes:

  • More reactions from the same volume
  • More space for low-concentrated sample
  • More space for primers and probes
  • Better stability
  • Best fit for multiplex reactions
This allows more flexibility with simpler sample preparation methods where DNA is not purified and concentrated, but stored in a transport buffer (mostly PBS-based) and added to the reaction with minimal pre-treatment steps.

Another important aspect is cross-contamination risk. It becomes really an issue when workflow becomes more intensive and usual safety methods are not enough. We recommend decreasing cross-contamination by using PCR mixes with UNG. 

If you’re doing a simpler sample preparation and input material contains different inhibitory substances, an inhibitor tolerant PCR reagent is of high importance. Read more about the remarkable inhibitor tolerance of SolisFAST® Probe qPCR range here: Taking the inhibitor tolerance to another level.

Check out the SolisFAST® Probe qPCR products:
References

[1] Multi-country monkeypox outbreak: situation update. World Health Organization. Available from: https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON390

[2] About Monkeypox. Centers for Disease Control and Prevention. Available from:  https://www.cdc.gov/poxvirus/monkeypox/about.html


[3] Laboratory testing for the monkeypox virus: Interim guidance. World Health Organization. Available from: https://www.who.int/publications/i/item/WHO-MPX-laboratory-2022.1

[4] Li, Y., Zhao, H., Wilkins, K., Hughes, C., Damon, I. K. (2010). Real-time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA. Journal of Virological Methods, 169(1):223–7.

[5] Schroeder, K., Nitsche, A. (2010). Multicolour, multiplex real-time PCR assay for the detection of human-pathogenic poxviruses. Molecular and Cellular Probes, 24(2):110–3.

[6] Maksyutov, R. A., Gavrilova, E. V., Shchelkunov, S. N. (2016). Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay. Journal of Virological Methods, 236:215–20.

[7] Li, D., Wilkins, K., McCollum, A. M., Osadebe, L., Kabamba, J., Nguete, B., et al. (2017). Evaluation of the GeneXpert for Human Monkeypox Diagnosis. Am J Trop Med Hyg, 96(2):405–10.

[8] Townsend, M. B., MacNeil, A., Reynolds, M. G., Hughes, C. M., Olson, V. A., Damon, I. K., et al. (2013). Evaluation of the Tetracore Orthopox BioThreat® antigen detection assay using laboratory grown orthopoxviruses and rash illness clinical specimens. Journal of Virological Methods, 187(1):37–42.

[9] European Virus Archive. Monkeypox virus. Available from: https://www.european-virus-archive.com/search/node/monkeypox.