Transcript Chapter 16 - Enterobacteriaceae

Clinical Virology:
Part One
Introduction
MLAB 2434 – Microbiology
Keri Brophy-Martinez
General Characteristics
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Obligate intracellular parasites
Identified by either cell culture OR
rapid tests from clinical specimens
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Enzyme Immunoassay (EIA)
Immunofluorescence
PCR/Nucleic Acid Probes
Structure of Viruses
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Contain a viral genome of
either RNA OR DNA
Genome can be double
stranded (ds) OR single
stranded (ss)
Protein coat (capsid)
Capsid + viral nuclei acid=
nucleocapsid
Genome + protein coat called
a virion
Some viruses have an
envelope
Classification of Viruses
DNA OR RNA
 Number of strands (ds or ss)
 Morphology
 Presence or absence of envelope
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Viral Reproduction
(Replication)
Unique to viruses
 Virus attaches to surface of
susceptible cell by specialized
structures on specific receptors on
the cell surface (ATTACHMENT)
 Virus enters cell by endocytosis
(fusion of viral membrane & cell
membrane) (PENETRATION)
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Viral Reproduction
(Replication) (cont’d)
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Inside the cell, virus loses protein
coat, releasing DNA or RNA
(UNCOATING)
Viral genome directs host cell to make
viral proteins and genome(ECLIPSE)
Virus-coded proteins and genome reassemble in host cell(ASSEMBLY)
New virions released by host cell lysis
OR budding from host cell
membrane(RELEASE)
Viral Reproduction
(Replication) (cont’d)
Specimen Collection and
Transport
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Viral shedding greatest during early stages
of infection, so specimens should be
collected as early as possible
Aspirates are best, but swabs are
acceptable if dacron or nylon is used
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Calcium alginate/ cotton swabs inhibit growth
of some viruses
Commercial viral transport systems
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Provide moisture, prevent contamination,
and preserve viral infectivity
Specimen Processing
Optimal to process viral cultures
immediately
 If impossible, store in refrigerator
up to 48 hours
 If longer, freeze at -70° C
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-20° C will cause crystal formation,
which disrupts host cells and results
in significant loss of viruses
Methods in Diagnostic
Virology
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Major methods to diagnose viral
infections
Direct detection of virus in clinical
specimen
 Serologic antibody assays to detect
viral antibodies
 Isolation of virus in culture
 Nucleic acid-based detection
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Direct Detection
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Advantages
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Rapid diagnosis
Detection of nonculturable viruses
No need for culture
Disadvantages
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Confined to specific virus
Dependent of specimen adequacy and
quality
Direct Detection (cont’d)
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Methods include
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Immunostaining/Immunofluorescence
Enzyme Immunoassay
Nucleic acid probes
Gene amplification assays- PCR
Electron microscopy
• looking for cell inclusions or cytopathic effects
on cells
Serologic Assays
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Indications for serologic assays
Diagnosis of infections with
nonculturable organisms like
hepatitis
 Absence of viral shedding
 Lack of available nucleic acid testing
 Determination of immune status (i.e.
rubella, etc.)
 Monitoring immunosuppressed or
transplant patients
 Epidemiologic studies
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Serologic Assays
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Problems with serologic assays
Measures host response rather than
detect virus
 Antibody-producing capabilities of
humans vary
 Antibody levels do not necessarily
correlate with acuteness of
infection
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Viral Isolation
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Three methods
Cell culture
 Animal inoculation
 Embryonated eggs
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Most cell cultures done for herpes
and genital and respiratory viruses
Cell Cultures
Once viruses are grown in cell
culture, cells are examined
microscopically for cytopathic
effects (CPE) on cells
 Some viruses, such as influenza, do
not cause CPE, so changes must be
demonstrated with
hemagglutionation or
immunofluoresence tests
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Types of Cell Culture
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Primary cell cultures
 Uses tissue from animals
 Seeded onto surface to form a monolayer
 Limited cell division
Diploid cell cultures
 Cells can divide up to 50 times
 Human neonatal lung (HNL) is an example
Continuous (heteroploid) cell cultures
 Cells are capable of unlimited cell division
 Derived from human cancer cells
Cell Cultures (cont’d)
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Advantages of cell culture
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Sensitive
Can identify broad spectrum of viruses
Disadvantages
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Time required for isolation and
identification
Viable organisms required
Specialized resources and personnel
needed
References
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Kiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory
Microbiology: A Practical Approach . Upper Saddle River, NJ:
Pearson Education.
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of
Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.
http://www.fifthdisease.org/general.html
http://www.idph.state.il.us/about/immunepics/measles.htm
http://www.idph.state.il.us/about/immunepics/mumps.htm
http://www.mc3cb.com/viruses.html