Diagnostic electron microscopy is still a timely and rewarding method

Abstract

Background: Parallel to its technical development starting in the 1930s, electron microscopy (EM) became an important tool in basic and clinical virology. First utilized in the rapid diagnosis of smallpox, it developed to a diagnostic routine in the early 1960s using the negative staining technique. EM was applied to infected cell-cultures and also to 'dirty' specimens including urine, feces, vesicle fluid, liquor. With the implementation of molecular biological and genetic techniques, the use of diagnostic EM decreased.

Objectives: (1) To give a perspective on future indications and possible uses by discussing the past and the present of diagnostic EM, (2) To describe the system of External Quality Assessment on EM virus diagnosis (EQA-EMV) established in 1994 by our laboratory and its achievements.

Study design: EQA-EMV is run to evaluate, to confirm and to improve the quality of diagnostic EM. Two different types of specimen are sent out: (1) prepared grids to assess and train the diagnostic skills of the participants, (2) stabilized virus particle suspensions to assess preparation efficiency.

Results: Diagnostic EM differs from other diagnostic tests in its rapidity and its undirected 'open view'. To emphasize these advantages, the indications for diagnostic EM are discussed, fundamental for a continuing future adaptation. Besides appropriate techniques, quality control measures are required to achieve and keep high diagnostic standards. The results from 6 years of EQA-EMV are presented.

Conclusions: In the history of diagnostic EM in virology, a change in use has been seen. Starting in the 1990s and coincident with the broad introduction of 'modern' diagnostic techniques, the number of EM diagnostic labs has decreased considerably--in spite of the obvious advantages of this technique. To guarantee the continuing performance of diagnostic EM in the future. EQA runs have to be performed as with other techniques in the diagnostic armament. The growing number of participants and participating countries indicates an interest in as well as a need for this program.

Fig. 1

Direct EM of (a) herpesvirus particles from a varicella vesicle and (b) parapoxvirus recovered from the skin ulcers of a diseased seal, demonstrating the principles of morphological diagnosis after negative staining and the advantages of using different stains. The membrane destroying and/or penetrating effect of 2% phosphotungstic acid helps to reveal the 100 nm herpesvirus capsid within the viral envelope. 1% uranyl acetate on the other hand, by its remarkable membrane stabilizing effects, reveals the surface detail of the brick-shaped poxvirus very clearly. Magnification, ×80 000; bar, 100 nm.

Fig. 2

Overview of the morphology of small round viruses, known to be causative agents of gastroenteritis. (a) calicivirus, named according to the very distinct capsid morphology with 32 deep surface indentations (Greek ‘calix’, cup, goblet). (b) astrovirus with their typical, star-like capsid symmetry (Greek ‘aster’, star). (c) Norwalk virus, barely displaying any surface detail, named after the place, where the first outbreak caused by this virus was described. (a)–(c) Negative staining using 1% uranyl acetate. Magnification, ×80 000; bar, 100 nm.

Fig. 3

Human polyomavirus from the urine of bone marrow transplant recipients. (a) Negative staining after ultracentrifuge enrichment (100 000 ×g, 1 h, 20°C) using 1% uranyl acetate. (b) Thin section EM of a sediment (see a) embedded in Epon showing an immune aggregate. (c) Thin section EM of diagnostic HEL cell cultures inoculated with urine after ultracentrifuge enrichment. A paracrystalline array of polyoma virus particles is displayed. Magnification, ×80 000; bar, 100 nm.

Fig. 4

Negative staining of purified cell culture isolates from (a) Ebola virus and (b) Marburg virus. The filamentous structures of Ebola virus generally longer than those shown by Marburg virus. Magnification, ×20 000; bar, 1000 nm.

Fig. 5

EQA map showing the participating countries in EQA 7 (distributed in February 1999) highlighted in dark gray. The development of the numbers of participants and participating countries, respectively, are shown in the lower left.