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External Quality Assurance Scheme in EM Virus Diagnostics (EQA-EMV): Discussion of Results from EQA-EMV No

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WHO Collaborating Centre for Emerging Infections and Biological Threats CONSULTANT LAB FOR DIAGNOSTIC ELECTRON MICROSCOPY OF INFECTIOUS PATHOGENS Dr. Michael Laue phone +49(0)30 18754 2675 Robert Koch Institute, Center for Biological Threats and Special Pathogens e-mail [email protected] Advanced Light- and Electron Microscopy (ZBS 4) web www.rki.de/cl-em Seestr. 10, D-13353 Berlin, Germany External Quality Assurance Scheme in EM Virus Diagnostics (EQA-EMV): Discussion of Results from EQA-EMV No. 31 (EQA-31) 24 April 2019 Dear participant of the EQA-31, We would like to thank you for your participation in the EQA-31, which was initiated in November 2018. If you have sent us a result sheet you will find attached a certificate, which states your diagnosis, the expected result and the number/percentage of correct diagnosis for each sample. Below, we discuss the overall outcome of EQA-31 and particular aspects worth to mention. General remarks and overall result EQA-31 comprised six virus suspensions (200 µl each), inactivated with 2% buffered paraformaldehyde and stabilized against bacterial growth with 0.02% sodium azide before shipment. The samples were shipped to 86 participants in 30 countries (Germany [33], other European countries [28], outside Europe [25]). In total, we have received 69 (80.2%) result letters. The complete panel was considered in the final evaluation because all of the six samples were correctly diagnosed by at least 5/6 reference labs. The correct diagnosis of all virus samples was achieved by 9/69 (13.0%) participants. The average of correct diagnoses of the whole panel is 68.6%, the average of all EQAs since 1994 is 72.2%. 5/6 correct results: 24 (34.8%) 4/6 17 (24.6%) 3/6 10 (14.5%) 2/6 05 (7.2%) 1/6 02 (2.9%) 0/6 02 (2.9%) 1 Remarks on the samples Sample 1: Species: Piscine novirhabdovirus. Synonym: Viral hemorrhagic septicemia virus (VHSV) (intended diagnosis: Rhabdoviridae) Family: Rhabdoviridae, Genus: Novirhabdovirus Correct diagnosis by ref. labs: 6/6 Following the 10th ICTV report on virus taxonomy “(https://talk.ictvonline.org/), the family Rhabdoviridae is now divided into 20 genera (in comparison to 6 in the 9th report). Species names have also changed. The ICTV committee seems to standardize all names in the manner “host (or disease) + genus”. In the case of sample 1 the Viral hemorrhagic septicemia virus is now termed Piscine novirhabdovirus. There are two bullet-shaped genera in this family infecting fish. Reporting Rhabdoviridae was correct. A diagnosis based on the genus level is not possible. 59/69 (85.5%) of the participants identified the rhabdovirus correctly. The sample had a high titer and virus particles were visible in large aggregates. Most of the particles were damaged and revealed the typical cross-striation of the helical nucleocapsid. Particles were bullet-shaped and therefore easily addressable to the family Rhabdoviridae. The mean examination time was short with about 14 min. Sample 2: Species: Orf virus. (intended diagnosis: Parapoxvirus) Family: Poxviridae, Subfamily: Chordopoxvirinae, Genus: Parapoxvirus Correct diagnosis by ref. labs: 6/6 Some minor changes in taxonomy according to the 10th report, nothing worth to mention, besides the fact that Orf virus still is termed Orf virus. Five participants seemed to be confused by the terminus “caprine pathogenic agent” in the EQA-letter and diagnosed Capripoxvirus. Sheeps and goats (plus further genera) are belonging to the subfamily Caprinae of the family Bovidae. These genera are the natural hosts of Orf virus. Participants should not take too much attention on the specimen information. It will help a little but not too much. Trust your eyes. Capripoxvirus is brick-shaped and orthopox-like. Look at the figure at the end of the report. In picture 2, the parapox- typic spiral coil around the ovoid particle is clearly visible. 60/69 (87%) reports were correct. The mean examination time was the shortest of the entire panel with about 13 min. 2 Sample 3: Species: Yellow fever virus (intended diagnosis: Flaviviridae) Family: Flaviviridae, Genus: Flavivirus Correct diagnosis by ref. labs: 5/6 Okay, we have to admit, that sample 3 was challenging because it contained a high concentration of small non-viral particles. Our “reference labs” and some other labs demonstrated their expertise. Good job. However, in total only 18/69 (26.1%) participants detected the virus. It is always a challenge to detect these small viruses. In this context our recommendation from the instruction file to use two different staining solutions with divergent characteristics becomes evident. In order to image flaviviruses accurately, high resolution stains like uranyl acetate or if you are not allowed to use uranyl acetate a stain like methylamine tungstate is preferred. They provide a better chance to visualize the lipid envelope. Phosphotungstic acid is in most instances a very good “allrounder”. You can use it to get a fast overview of the grid and PTA matches for most viruses very well. With PTA you will detect small particles but in the case of very small viruses you might not get enough information of the fine surface details, i.e. you have less resolution. However, in the figure at the end of the report, you will find a nice “lucky shot” of the Yellow fever virus with PTA staining. The average examination time was high with about 44 min and some participants got really “mad” about it with hours of hours time investment. Among the Flaviviridae, virions of the genus Pegivirus seem not to be visualized until now. If someone has information about viruses of this genus, we would be happy if you could share it with us. Sample 4: Species: Measles morbillivirus (intended diagnosis: Paramyxoviridae or Pneumoviridae, both were accepted, see below) Family: Paramyxoviridae, Subfamily: Orthoparamyxovirinae, Genus: Morbillivirus Correct diagnosis by ref. labs: 6/6 The virus family Pneumoviridae (formerly subfamily Pneumovirinae within family Paramyxoviridae) is difficult to differiantiate from paramyxoviruses by negative staining. Pneumovirus group specific is a second matrix protein layer (M2-1), which is lying unterneath the lipid envelope and the first matrix layer. To analyze this second layer we would need perfect conditions (concentration, purity, a lot of damaged particles and time) but in diagnostic samples we never have perfect conditions and rarely enough time. So, in future we will accept both 3 diagnoses. Reporting Paramyxoviridae or Pneumoviridae is the best choice. Paramyxoviruses are such divergent in shape and size that we always receive reports of orthomyxovirus as well. Detection of released ribonucleoprotein is necessary for the correct diagnosis of para- or pneumoviruses, see picture 4 in the figure at the end of this report. Naming of the species has also changed and the family Paramyxoviridae has now splitted from two into four subfamilies. 58/69 (84.1%) reports were accepted as correct. The mean examination time was about 19 min. Sample 5: Species: Infectious pancreatic necrosis virus (IPNV) (intended diagnosis: Birnaviridae was accepted, Aquabirnavirus due to given host information) Family: Birnaviridae, Genus: Aquabirnavirus Correct diagnosis by ref. labs: 6/6 No changes in the taxonomical structure of the virus group (unbelievable). 76.8% (53/69) of success was very satisfying. As expected some labs found an adenovirus or a reovirus presumably because birnaviruses are rarely seen and particles of family Birnaviridae are nicely icosahedral (see picture 5). Measuring helps a lot to avoid a mis-diagnosis. Adenoviruses are much larger with triangles on the surface and sometimes fibers are visible. Reoviruses appear rounder in shape and larger as well. The mean examination time was about 27 min. Sample 6: Species: Chikungunya virus (intended diagnosis: Togaviridae, Alphavirus) Family: Togaviridae, Genus: Alphavirus Correct diagnosis by ref. labs: 6/6 No changes in the taxonomical structure of the virus group. We are proud that we could include for the first time a togavirus into the EQA scheme. 38/69 (55.1%) of the participants got the clue. With a mean examination time of about 36 min the diagnosis took a while. Besides the correct diagnosis many other “ideas” were reported. This was probably due to the particle size of 70 nm which fits in the particle size range of other virus families, reoviruses for instance. However, if measurements would have been conducted and considered, mis-diagnoses like herpes-, lenti-, polyoma-, calici-, bunya-, corona-, orthomyxo- or flavivirus (16 times!) would have been avoided. Please, allways measure particles of interest (with a magnification-calibrated microscope) and use a virus image atlas or the VirusExplorer if you are not sure about something new. 4 Concluding remarks Response and outcome of EQA-31 are again very satisfying and it is always a pleasure to conduct the EQA scheme. Regular participants may have recognized that the attendance numbers are decreasing over time. We like to encourage you to support us. Please recommend the EQA to peers in the field. Another point to mention is that it gets more and more complicated to ship the panel to targets outside the Schengen (EU and associated) state area. Shipping time can last up to 2 months or more and sometimes transport fails at all due to harsh customs restrictions or other reasons. The only way to avoid shipping delay is to mandate worldwide operating logistic companies with the transport. This is costly but usually works well. After having commissioned them, they will contact us for doing the paper work, which is annoying but not avoidable. We are conducting the EQA scheme free of charge and do not have the funding to pay the special transportation fees charged by an ordered transport. So, if you want to order a transport for your next EQA-samples, get in touch with us.
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