electron microscopy in diagnosis of infectious … microscopy in diagnosis of infectious diseases...
TRANSCRIPT
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Electron Microscopy in Diagnosis of Infectious Diseases
Sara E. Miller, Ph. D.
Department of Pathology Duke University Medical Center
Durham, NC 27710 [email protected]
(919) 684-9141
Duke Medicine Society for Ultrastructural Pathology
January 29, 2013
A
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Outline Part 1
A. Virology 1. Advantages of using EM in diagnostic virology 2. Limitations of using EM in diagnostic virology 3. Workarounds 4. Virus structure
a. In negative stains b. In thin sections
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Outline Part 1
B. Other organisms 1. Bacteriology 2. Mycology 3. Photology (algae) 4. Protozoology
Questions/Discussion
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Outline Part 2
C. Virus look-alikes 1. Examples of confusing things in fluids 2. Examples of cell organelles that resemble
viruses in tissues
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Outline Part 2
D. Real cases 1. Examples of organisms diagnosed from patients 2. Quiz for fun
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Outline Part 1
A. Virology 1. Advantages of using EM in diagnostic virology 2. Limitations of using EM in diagnostic virology 3. Workarounds 4. Virus structure
a. In negative stains b. In thin sections
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A. Virology
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Advantages of EM in Virus Diagnosis/Identification • Fast • Does not require living organisms • Does not require special reagents (antibodies, nucleic acid standards, protein standards) • Can visualize other organisms: odd agents, odd locations • Works when other modalities are not enough (mutant genomes may not react in PCRs) • No false positive results (cross-reactions with similar things) • What you see is the real thing
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Limitations of EM in Virus Diagnosis/Identification
• Less sensitive than some other tests
• Requires expensive instruments (EM)
• Requires virology knowledge
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Ways To Get Around Limitations • Ultracentrifugation • Antibody concentration • Confocal microscopy of wet tissue slabs • Multiple tissue locations • Semi-thin sections of epoxy-embedded tissues • Where to look in tissues: inflammation, nucleated cells, necrosis edge, unusual ultrastructure for the tissue type), syncytia
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Virus Terminology
Naked Enveloped
Nucleocapsid
Capsid
Envelope
NA NA
NA
Virion (complete virus)
] ]
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Identification of Viruses in Fluids
70-90 nm
Naked: Icosahedral
Enveloped: Pleomorphic
30-35 nm
45-55 nm
70-90 nm
150-300 nm
Miller SE. 1991. In de la Maza LM, Peterson EM (eds), Medical Virology, Vol. 10. Irvine, CA. pp 21-54.
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80 nm x 300 nm 80 nm x 1400 nm
200 nm x 400 nm
Enveloped: Not Pleomorphic
Identification of Viruses in Fluids, Con’t.
Miller SE. 1991. In de la Maza LM, Peterson EM (eds), Medical Virology, Vol. 10. Irvine, CA. pp 21-54.
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Polyomavirus from Urine Enteric Viruses
Influenzavirus
G
100 nm
Negative Staining of Naked and Enveloped Viruses
Rotavirus
Coronavirus
Calicivirus
Adenovirus
H
Astrovirus
Picornavirus
100 nm
100 nm
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Negative Staining of Spherical and Helical Nucleocapsids
virus
100 nm
Herpesvirus
nc
nc
virus
virus 100 nm
Measlesvirus
nc
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Naked (Icosahedral) 3 size ranges Paracrystalline arrays
Enveloped (Pleomorphic) Membrane-associated “Smooth” Fringed Nucleocapsids Spherical Helical
Identification of Viruses in Tissues
Location in cells DNA--nucleus RNA--cytoplasm
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DNA Viruses: Usually produced in the nucleus
RNA Viruses: Usually produced in the cytoplasm
1.O µm
Reovirus µm
Adenovirus
N
N
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Nuclear Membrane Budding
1 µm
100 nm
Plasma Membrane Budding
HTLV
nc
Herpesvirus
Vesicular Budding
Budding
N
N 100 nm
100 nm
N virus
Hantavirus
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Helical Nucleocapsids
Spherical Nucleocapsids
N
100 nm
100 nm
µm 1
Measlesvirus
Herpesvirus
N nc
nc
nc nc
nc virus
virus
virus
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Bioterrorism: the intentional release or dissemination of biological agents such as bacteria, viruses, or toxins to create disease and fear.
EM in Surveillance of Bioterrorism and Emerging Diseases
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Laboratory Response Network (LRN)
• Collaboration: FBI & Association of Public Health
Laboratories • Started: August, 1999 • Includes: State & local public health labs,
veterinary, agriculture, military, environmental, & water- & food-testing labs
• Function: rapid testing, timely notification, & secure results reporting from public health emergencies (biological/chemical terrorism & emerging diseases)
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LRNs and Electron Microscopy
• About half the LRNs are paired with electron
microscopy laboratories. • The Duke Electron Microscopy Service (DEMS) is
the EM partner of the NC State Public Health Laboratory in the NC LRN.
• If asked to process biohazard sample, read Dos and Don’ts article.
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References for Protocols: Negative Staining Electron Microscopic Protocol for Rash Illness. http://www.bt.cdc.gov/labissues/ Then click on title above. Electron Microscopy for Rapid Diagnosis of Emerging Infectious Agents. http://wwwnc.cdc.gov/eid/article/9/3/02-0327_article.htm Bioterrorism and electron microscopic differentiation of poxviruses from herpesviruses: dos and don’ts. Ultrastruc Pathol. 2003;27:133-140. Modern uses of electron microscopy for detection of viruses. Clin Microbiol Rev. 2009;Oct;22(4):552-63. doi: 10.1128/CMR.00027-09. Review Detection and identification of viruses by electron microscopy. J Electron Microsc Tech 4:265-301;1986.
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Smallpox virus • Easily disseminated • Easily weaponized • High mortality rate, survivors badly scarred • Previously mass-produced • Believed to have been distributed after USSR collapse Hemorrhagic fever viruses • Filoviruses (Marburg, Ebola) • Arenaviruses (Lassa, Machupo)
Class A Viral Agents of Bioterrorism
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Because of ID speed, EM is on the front line in bioterrorism surveillance (e.g., smallpox).
Poxvirus lesions are most likely to be confused with varicella-zoster virus (VZV) (a herpesvirus) lesions.
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Palmer E, Martin ML. CRC.
EM in Surveillance of Bioterrorism
Orthopoxvirus Parapoxvirus Herpesvirus
100 nm
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Poxvirus
N
Immature Particles Mature Particles
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Herpesvirus
100 nm
1 μm
N
N
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EM in Surveillance of Emerging Diseases
Viral emerging diseases identified first by EM • Parvovirus B-19 • Monkeypox • SARS coronavirus • Metapneumovirus • Morbilivirus • Nipah virus
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Costs (11/02 – 7/03): 774 Deaths, 9.6% fatality (China, Hong Kong, Taiwan, Canada) $11 billion (Asia alone)
EM was crucial in identifying the coronavirus in the SARS outbreak.
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Courtesy of Cynthia Goldsmith, CDC, Atlanta.
Thin Section of SARS Coronavirus
100 nm
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EM was crucial in identifying the monkeypox virus outbreak
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Thin Section of Monkeypox Virus Infected Cell
Negative Stain
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Summary: Identification of Viruses
• In fluids (negative stains) o Naked icosahedral: size, surface structure o Enveloped: size, surface structure of core/envelope
• In tissues (thin sections) o Naked o Enveloped (usually budding) o Location: nucleus/cytoplasm