Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must?
Abstract
:1. Introduction
2. Orthopoxviruses (OPV), Herpesviruses and Other Agents
3. Smallpox (Variola)—The Disease
4. Diagnosis of Vesicular Skin Rashes
4.1. Unique Advantages of DEM in Rapid Viral Diagnosis
4.2. Specimen Collection and Preparation
Collecting Diagnostic Fluids from Vesicles
- Aspirate fluid from three lesions into a small tuberculin-type syringe with a fine needle (29 gauge is ideal). Carefully re-cap the syringe and place it into a transport container. Cave: Recapping syringes is strictly forbidden in the US for safety reasons (unless it is one-handed or done with a device to hold the cap).
- Open the surface of a vesicle using a sterile (injection) needle. Collect fluid into a fine glass tube by capillary attraction and close the tube at both ends with dental wax. The wax is removed later in a Laminar Flow Safety Cabinet and the fluid is carefully expelled onto a sheet of ParafilmTM using a small rubber bulb on the “clean” end of the capillary.
- Open the vesicle as in 2 above. Press the middle of a sterile glass light microscopic slide onto the fluid, remove the slide, let it dry, and mark it on the reverse where the droplet has dried to make finding the sample easier. Place the slide in a Petri dish for transport. To protect the dried fluid during transport, two matchsticks are placed as spacers at the ends of the sample slide which is then covered with another plain slide. Both slides are then bound together using an elastic band at each end.
- With an open ulcerated lesion or when the base of a vesicle is uncovered, touch a microscope grid briefly onto the base holding it with fine-pointed forceps. Place the grid with specimen side uppermost onto a filter-paper disc placed in a Petri dish. Cover before transporting to the laboratory.
4.3. Specimen Support Grids for DEM
4.4. Negative Staining of a DEM Sample
- To avoid cross-contamination use a different pair of forceps for each specimen. Always disinfect and clean the forceps carefully immediately afterwards.
- The washing steps are helpful in getting an even distribution of stain on the grid. However, each washing step will appreciably reduce the number of particles on the grid.
- Before staining with UAc, the grid with adherent specimen material must be washed on 3–5 droplets of distilled water to remove interfering phosphate ions. Successful NS with PTA does not require extensive washing; washing on a single droplet will suffice.
- While checking the first grid in the TEM, the remaining specimen, the washing and stain droplets are left on the ParafilmTM, protected from dust and drying in the wet chamber. This helps to shorten preparation time in case further preparations, possibly stained differently, are needed.
- Viruses and other biologicals will concentrate at the interface between the air and the sample fluid [103]. Letting the sample droplet remain untouched for a few minutes (or even longer, e.g., overnight at 4 °C in the refrigerator, if found necessary) before the grid is placed onto it for adsorption can help with low-concentration samples.
- With sample volumes below 5 µL the sample may be placed directly onto the grid´s surface for adsorption.
- While PTA staining tends to make biological structures more labile and porous, UAc is both a stain and a fixative [104]. When one stain does not result in a satisfactory preparation, the other usually does. Therefore, with unknown samples it may be advisable to use both stains in parallel. Preparations stained with either stain tend to deteriorate in the course of a fortnight. A loss of fine structure and the appearance of larger stain crystals (“grain”) can be avoided by storing “valuable” grids in vacuo in a desiccator containing some phospho-pentoxide as a desiccant. As well as these two common stains, a number of alternatives, e.g., ammonium molybdate, sodium silico-tungstate and uranyl formate have also been used successfully [90,98,105]. All three excel by a very fine “grain” and ammonium molybdate in addition by a well-balanced contrast.
4.5. Biological Safety in DEM
5. Limitations of DEM
5.1. Cost and Availability of a Specific DEM Laboratory
5.2. Too Few Particles in the Specimen
5.3. Lack of Dedicated and Experienced Staff for DEM
5.4. Low Sample Through-Put by DEM
6. Conclusions
7. What is the Future for DEM?
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
BPXV | Buffalopox virus |
CAM | Chorio-Allantoic Membrane of fertile hen’s egg, used for isolating pox and herpes viruses |
CDC | Centers for Disease Control & Prevention, Atlanta, GA, USA |
CMLV | Camelpox virus |
CPXV | Cowpox virus |
DEM | Diagnostic Electron Microscopy |
DNA | DeoxyriboNucleic Acid |
ELISA | Enzyme-Linked Immunosorbent Assay (often abbreviated to EIA) |
EM | Electron Microscope or Electron Microscopy |
GLP | Good Laboratory Practice |
HSV | Herpes Simplex Virus |
MC | Molluscum Contagiosum |
MCV | Molluscipox Virus |
MERS | Middle East Respiratory Syndrome |
MPXV | Monkeypox virus |
NAT | Nucleic Acid Amplification Techniques |
NS | Negative Staining |
NS-EM | Negative Staining Electron Microscopy |
OPD | Out-Patients Department |
OPV | OrthoPoxVirus |
PCR | Polymerase Chain Reaction |
PPV | ParaPoxVirus |
PTA | Potassium phosphoTungstic Acid |
SARS | Severe Acute Respiratory Syndrome |
TEM | Transmission Electron Microscopy |
TS | Thin Section |
TS-EM | Thin-Section Electron microscopy |
UAc | Uranyl Acetate |
VACV | Vaccinia virus, used for prophylaxis against smallpox |
VARV | Variola virus, cause of Smallpox |
VZV | Varicella Zoster Virus |
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Micro-Organism, Agent or Condition | Disease | Lesion Appearance * |
---|---|---|
Orthopoxviridae (OPV) Variola major virus Variola minor = Alastrim Man is only known host, currently eradicated as a human disease | Smallpox Alastrim | Generalised vesicular rash with large deep-seated vesicles all at the same stage, dimpled at the centre developing into pustules and crusting over later. Centrifugal distribution, including soles and palms. May be modified by previous vaccination |
Other OPV: Vaccinia virus, buffalopox, cowpox, monkeypox, camelpox | Various animal diseases, occasionally transmitted to man | Usually single vesicular or papular lesion, developing into ulcer, and crusting later Lesions larger, up to 1cm in diameter, may not be clearly vesicular |
Parapoxviridae (PPV) of goat, sheep: orf; cattle: paravaccinia, pseudocowpox | Animal diseases transmitted to man as Orf, Pseudocowpox (Milker’s nodules) | Large (up to 1 cm) nodular with little vesicular fluid, developing into an ulcer, crusting later May not be clearly vesicular |
Molluscipoxvirus (MCV) specific for man. There is also animal specific MCV | Molluscum contagiosum warty lesions–may be multiple, may be passed as a sexually transmitted disease. Auto-inoculation may spread the lesions | Solid, firm, wart-like tumours: dome-shaped or flat. Pearly or flesh-coloured nodules with a depression on the top. Not clearly vesicular but contain waxy sacs packed with virus particles |
Herpes varicella zoster (VZV) | Chickenpox, usually in childhood or Shingles (herpes zoster = recrudescence of previous varicella) | Generalised or scanty vesicular rash, mostly on head and trunk becoming pustular and crusting later. Lesions smaller, frailer and less deep-seated than smallpox, can be easily ruptured. Differ in size and stage of development Shingles: similar lesions but confined to the distribution of one or more sensory nerves. |
Herpes simplex virus HSV-1, HSV-2 | “Cold sores”, usually limited to a few localised lesions, usually on the upper lip. Very occasionally a herpes encephalitis. Herpes simplex may be sexually transmitted | Limited recurrent vesicular lesions with prodromal tingling Lesions smaller and less deep-seated than smallpox, crusting later. |
Enterovirus and other small spherical RNA-containing viruses | Hand-foot-and-mouth disease | Aphthous oral ulcers and small vesicular lesions 2–4 mm in diameter on the hands and feet, can also generalize. |
Anthrax, Bacillus anthracis | Cutaneous Anthrax | Single or small number of large vesicles later, surrounding a dark central crust (“Malignant pustule”) |
Treponema pallidum | Primary and secondary syphilis | Single red papule 0.5 to 2 cm in diameter developing into ulcer with an indurated margin and exudate |
Drug-induced rashes | A variety of rashes: exanthematous pustulosis, Erythema multiforme | No specific micro-organisms present |
Scabies and insect bites | Variety of single or multiple quasi-vesicular lesions | No specific micro-organisms present |
Contact dermatitis | Symptomatic toxic-dermatitis | No specific micro-organisms present |
Genus | Members, Species | Disease in Healthy Men | Natural Host | Appearance in DEM and Size |
---|---|---|---|---|
Orthopoxvirus (OPV) | Variola virus (VARV) | Smallpox Variola major Variola minor | Man only | Brick-shaped virions, 250–350 nm × 200 nm with an irregular array of 10–15 nm surface “protrusions” (threads). |
Vaccinia virus (VACV) | Vaccination = local, self-limiting lesion | endemic in cattle and buffaloes in India and Brazil | ||
Cowpox virus (CPXV) | Scanty vesicular rash developing into ulcer | Rodents transmitting CPXV to cattle, cats and other mammals | ||
Monkeypox virus (MPXV) | Vesicular rash, similar to smallpox | Squirrels, non-human primates | ||
other OPV: camelpox, buffalopox | Single or multiple vesicular-pustular lesions developing into ulcer | Various * | ||
mousepox (ectromelia) and several others, some unclassified | no known disease in man | |||
Parapoxvirus (PPV) | Orf (ecthyma contagiosum) | Single tender nodule developing into ulcer 10–15 mm in size | Sheep, goats | Oval virions: 250–300 nm × 150–180 nm with long, spiral surface threads |
Pseudocowpox, Bovine papular stomatitis | see Orf | Cattle | ||
Molluscipoxvirus (MCV) | Molluscum contagiosum virus (MCV) | Single or multiple papules developing into pink fleshy “warts”, often with umbilicated centre | Man only | Brick-shaped virion, short threads: Very similar to OPV |
Yatapoxvirus | Tanapox | Single or multiple firm nodules | Non-human primates | Brick-shaped, very similar to OPV |
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Gelderblom, H.R.; Madeley, D. Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must? Viruses 2018, 10, 142. https://doi.org/10.3390/v10040142
Gelderblom HR, Madeley D. Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must? Viruses. 2018; 10(4):142. https://doi.org/10.3390/v10040142
Chicago/Turabian StyleGelderblom, Hans R., and Dick Madeley. 2018. "Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must?" Viruses 10, no. 4: 142. https://doi.org/10.3390/v10040142
APA StyleGelderblom, H. R., & Madeley, D. (2018). Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must? Viruses, 10(4), 142. https://doi.org/10.3390/v10040142