Electron Microscopy for Rapid Diagnosis of Infectious Agents in Emergent Situations

Specimen Collection

Successful investigation of any outbreak or novel case starts with specimen collection. Insufficient, improper, or inadequate sampling may delay or prevent identification of a causative agent. Sufficient sampling requires identification of, and sampling from, all areas where infection may have been established. Fecal samples are ideal for investigating gastroenteric episodes, as are lesion fluids or smears from skin lesions of possible viral origin.

A major cause of insufficient sampling can be failure to collect acute-phase sera from affected case-patients and more importantly, their contacts, who might well be asymptomatic. First, the existence of a blood-borne pathogen may not be evident when examining unexplained cases, as demonstrated by the difficulty identifying HIV^[27] and hepatitis C virus infections, and associating human parvovirus B-19 with Fifth disease.^[16] Second, acute-phase sera are essential for demonstrating seroconversion to a suspected agent. Third, clinical symptoms may be caused by an immune response to an infection that has resolved by the time they appear. However, specimens from apparently uninfected contacts of patients with acute cases may contain the agent involved.^[16] Convalescent-phase sera collected from case-patients 4-6 weeks after onset of illness are also powerful diagnostic reagents. If no agent has been identified by standard virus detection procedures (e.g., electron microscopy, tissue culture, immunoassay, or nucleic acid amplification techniques), these serum samples may be used to detect the causative agent,^[28] while matched acute:convalescent-phase serum pairs collected at least 2 weeks apart may be used to demonstrate a significant rise in specific antibody among cases by immuno-electron microscopy (Figure 2).^[7] Infectious agents may also be identified in cerebrospinal fluid, lesion crusts, nasopharyngeal washes, saliva, tears, urine, and biopsied tissue specimens.^[29] However, low viral load, sampling difficulties, or both may reduce the effectiveness of rapid electron microscopic diagnosis on these later types of specimens without initial tissue culture amplification, as observed in Nipah virus studies.^[6]

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Association of human parvovirus B-19 with erythema infectiosum by immuno electron microscopic.

Safety concerns, miscommunication between infectious diseases specialists and staff who collect samples, or inadequate training may result in improper sample collection. Although swab samples placed into viral transport media may allow nucleic acid amplification techniques or culture of nonfastidious agents to be carried out, such specimens are not very conducive to successful rapid electron microscopy diagnosis of lesion exudates because of dilution effects and interfering components. Several effective ways of collecting lesion fluids exist.^[8] A method readily available to the physician or in a hospital ward is collection into the barrel of a 26-gauge needle attached to a tuberculin syringe. A fresh lesion is unroofed or the beveled surface of the needle is placed against the base of an open lesion, and fluid is aspirated into the barrel. After capping, the sample may be transported directly for rapid electron microscope diagnosis (Figure 3A). Alternatively, coated electron microscope specimen grids may be lightly touched directly to the vesicle fluid, lesion base, or both; allowed to air dry; and transported directly for examination (direct touch preparation) (Figure 3B). Because repreparing the sample with direct touch preparations may not be possible, at least two grids should be obtained when the specimen is collected. For safety and containment of hazardous infectious materials, the syringe or grid should be placed in a rigid sterile container, e.g., conical 15-mL centrifuge tube or Beem capsule (Beem Co., Bronx, NY), sealed with Parafilm (American National Can Co., Greenwich, CT), and the outside of the tube washed with 0.5% sodium hypochlorite (10% household bleach) before transport (Figure 3). Safety regulations usually require further packaging of the sample inside a second container.

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Three methods for efficient collection of vesicular and blister fluids for diagnostic electron microscopy.

In the late 1940s, direct touch preparations from skin lesions were prepared in North Africa and sent to Toronto, Canada, where they were examined successfully for smallpox virus for up to 4 months after collection.^[11] In another comparative study in Winnipeg, Canada, which used matched lesions, we observed an average increase of 10.2:1 in the number of virions visualized by direct touch as opposed to needle aspirate preparations, and the ratio was >1.0 in 92% of total cases examined (n=12; p<0.02; [Wilcoxon signed-rank test]). We observed no difference in the number of positive identifications or homogeneity of virion distribution on the grid between these two methods. Lesion smears on glass slides may also be used effectively for both electron microscopy and immunofluorescent microscopy examination (Figure 3C). Smears, i.e., dried down vesicle fluids, are especially effective when syringes and electron microscopic grids are not available. Both direct touch and smear preparations are useful when specimens must be transported some distance for electron microscopic examination.

The collection of lesion exudates as swab samples placed in viral transport medium is less effective. A change in specimen collection protocols in 1995, from direct touch/lesion aspirates to swab specimens in transport medium, has resulted in a decline in successful identification of virions in lesion specimens in Winnipeg from 62% to 75% to approximately 10% (Hazelton, unpub. data). While complete fecal samples are preferable, collecting rectal swab samples for diagnosis of gastroenteric agents may be necessary. These swab samples should be placed in capped conical centrifuge tubes with 0.2 mL sterile, distilled water, sealed with Parafilm, and sent for electron microscopic diagnosis. Lesion crusts should also be placed in sterile conical tubes. The addition of any liquid medium to lesion crusts, cerebrospinal fluid, nasopharyngeal washes, saliva, tears, and urine will not assist the electron microscope laboratory. Tissue biopsy samples in buffer without fixatives should be stored at 4°C and sent directly to both an electron microscope facility and a viral identification laboratory for rapid electron microscopy and other diagnostic testing. Fixation may interfere with antibody binding and thus preclude infectivity tests and successful application of any immunoelectron microscopy.

Finally, failure to collect an adequate volume of sample will limit the tests that may be used and the ability to successfully identify causative agents. Lesion fluids are deceiving. For example, samples containing poxvirus or varicella zoster virus that appear to have no material drawn into a needle barrel (Figure 3A) or attached to a grid may still contain numerous virions. When possible, at least 1 g of fecal material should be collected into a commercial stool collection vessel. A minimum of 5.0 mL of blood should be collected into tubes without anticoagulants. When a special interest in the case or outbreak occurs, large samples may provide reagents for later testing. All samples should be immediately sent for rapid electron microscopic diagnosis, with storage at 4°C if possible. Dried smears may be stored and transported at ambient temperature. Under no condition should samples be frozen for storage and transport before receipt at the diagnostic facility.^[30]

Emerging Infectious Diseases. 2003;9(3) © 2003 Centers for Disease Control and Prevention (CDC)

Cite this: Electron Microscopy for Rapid Diagnosis of Infectious Agents in Emergent Situations - Medscape - Mar 01, 2003.