Diagnostic electron microscopy (DEM) was an essential component of viral diagnosis until the development of highly sensitive nucleic acid amplification techniques (NAT). The simple negative staining technique of DEM was applied widely to smallpox diagnosis until the world-wide eradication of the human-specific pathogen in 1980. Since then, the threat of smallpox re-emerging through laboratory escape, molecular manipulation, synthetic biology or bioterrorism has not totally disappeared and would be a major problem in an unvaccinated population. Other animal poxviruses may also emerge as human pathogens. With its rapid results (only a few minutes after arrival of the specimen), no requirement for specific reagents and its "open view," DEM remains an important component of virus diagnosis, particularly because it can easily and reliably distinguish smallpox virus or any other member of the orthopoxvirus (OPV) genus from parapoxviruses (PPV) and the far more common and less serious herpesviruses (herpes simplex and varicella zoster). Preparation, enrichment, examination, internal standards and suitable organisations are discussed to make clear its continuing value as a diagnostic technique. in childhood, it was clear that chickenpox was what he now had. No national alert was necessary, with all the widespread anxiety that would have followed. The speed and certainty of the diagnosis by EM offered a lesson that still has relevance. Four valuable lessons can be drawn from this episode: (1) That a useful diagnosis can be made in minutes from the arrival of the specimen; (2) Diagnostic EM (DEM) required no specific reagents (such as primers or antisera) or special equipment. All that is required was an electron microscope, microscope support grids, stain and a competent virologist familiar with the appearance of relevant viruses; (3) That seeing is believing-knowing what the causative virus looks like is a useful confirmation for any other tests added later; (4) That other, more serious, causes could be discounted. With concerns over the possible re-emergence of poxvirus infections, either through the spread of existing animal viruses to susceptible humans [1] [2] [3] [4] [5] , survival and escape of old material [6-8], molecular manipulation and synthetic biology [9] or even bioterrorism [10-12], there will be a need to provide accurate and quick diagnosis. This paper presents the possibilities and advantages of DEM and how DEM can help with organised future preparedness. Viruses 2018, 10, x 8 of 30 at Library of the RKI for help with the acquisition of published information, and Lars Möller, ZBS 4, RKI for his kind help to prepare an online-version of our manuscript. Thanks are also due to Anna-M. Eis-Hübinger, Bonn, and Bernhard Pfeiff for information on the fatal CPXV case and kindly providing images. We acknowledge the generous help by Gudrun Wibbelt, IZW Berlin, Pierre Formenty, WHO Geneva, and Cynthia Goldsmith, CDC, with the acquisition of clinical images of CPXV and the MPXV zoonoses respectively. Hermann Meyer, Munich, supplied hg readily with inactivated MPXV samples from the DRC. For supplying diagnostic PPV samples repeatedly, we thank Klaus Eisendle, Bolzano and Friedrich A. Bahmer, Bremen. Thanks are finally due to the many clinical colleagues we had the pleasure to work with over some 60 years all together.
Output: viruses Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must?