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CP Board Review - Microbiology > Virology > Flashcards

Virology Flashcards

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1
Q

Introduction – Viruses

A

Subcellular pathogens
 DNA and RNA genomes
 Cause a wide variety of disease
 By far the most rapidly changing area of clinical microbiology

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2
Q

Classification of Viruses

A 
Genome: RNA (14 families) and DNA (7 families); ss, ds, linear, circular, single or multiple segments
 Enveloped and non-enveloped
 Morphologic
– Icosahedral
– Helical
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3
Q

DNA Viruses

A

 Double-stranded linear genomes except as noted.

 Parvoviridae; ssDNA
– B19

 Hepadnaviridae; enveloped
– Hepatitis B virus

 Polyomaviridae; circular genome
– JC & BK viruses

 Papillomaviridae; circular genome
– Human papillomavirus

 Adenoviridae
– Many serotypes

 Herpesviridae; enveloped
– EBV, CMV, VZV; HSV; HHV 6-8.

 Poxviridae ; enveloped
– Smallpox, vaccinia, molluscum contagiosum

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4
Q

RNA Viruses

A

 Retroviridae; enveloped
– HIV, HTLV,

 dsRNA viruses
– Reoviridae - Rotavirus

 Minus-strand ssRNA viruses
– Paramyxoviridae; enveloped - Parainfluenza, measles, mumps, RSV

– Rhabdoviridae; enveloped - Rabies virus, VSV

– Filoviridae ; enveloped - Ebola, Marburg

– Orthomyxoviridae; enveloped - Influenza

– Bunyaviridae; some enveloped - La crosse, hantaviruses, CCHF, RVF

– Arenaviridae; enveloped - Lymphocytic choriomeningitis virus

 Plus-strand ssRNA viruses
– Picornaviridae - Enteroviruses, rhinoviruses, hepatitis A virus

– Caliciviridiae - Noroviruses, hepatitis E virus

– Astroviridae - Human astroviruses (gastroenteritis)

– Coronaviridae; enveloped - SARS, other human coronaviruses

– Flaviviridae; enveloped - Yellow fever, hepatitis C virus

– Togaviridae; enveloped - WEE, EEE, rubella

 Others
– ssRNA satellite agent: hepatitis delta virus
– prions

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5
Q

Viral Diagnostic Methods

A

 Viral culture methods
 Antigen detection methods
 Nucleic Acid testing methods
 Viral Serology / Antibody Detection

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6
Q

Viral Culture

A

 Requires
–Permissive cells
–Means of detecting growth
–Means of identifying virus

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7
Q

Types of Viral Culture

A

 Viral tube culture

– Cell types
 Primary cells, e.g. monkey kidney cells,
 Diploid cell cultures, e.g. human foreskin fibroblasts, human embryonic lung
 Permanent cell lines, e.g. HeLa, A549, MRC-5, HEp-2

– Detection by seeing cytopathic effect (CPE) in 1-20+ days

– Identification with specific Fluorescent Antibody (FA) testing

– Even in the molecular era, viral culture is still a primary way of detecting novel and unexpected viruses

 Shell vials – targeted cultures
– Inoculation of cells on a cover slip in a small tube, a ‘shell vial’
 Centrifugation often improves yield
– Short incubation (24-48 hours)
– FA or other stain for specific target viruses

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8
Q

Viral Tube Cultures – Detection

A

Cytopathic Effect (CPE)
– Early; rounding of cells
– Later: cell death, ‘plaques’
– CPE characteristics (time, morphology) relate to possible viral etiologies

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9
Q

Viral Conventional Culture – Cells

A 
 Different viruses, different cells
 Limitations
– Slowgrowing viruses ex. CMV
– Nongrowing viruses ex. HBV, HPV
– Loss in transport
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10
Q

Viral Culture – Types of CPE

A

this is table need to type in

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11
Q

Shell Vial Culture

A

 Culture 1-2d on coverslip
 Stain for specific target viruses, esp. early antigens
 Slow-growing viruses e.g. CMV
 Respiratory viruses
– Cell mixes and antibody cocktails
 Other samples with defined targets, e.g. skin lesions
 Highly sensitive and specific
 Relatively rapid
 Detects only the defined viruses you’re looking for.

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12
Q

Viral Antigen Detection

A

 A variety of methods
– DFA
– Instrumented EIAs (esp. for hepatitis)
– Rapid tests for respiratory and GI viruses
 Usually faster than culture
 Some are automated; some are point-of care
 Usually insensitive relative to culture or molecular methods

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13
Q

Viral DFA

A 
 Slide made directly from sample
– Stained with fluorescent antibody
 Skin lesions: HSV / VZV
 Respiratory samples:
Influenza, assorted respiratory viruses
 High skill required, can approach sensitivity of culture
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14
Q

Rapid Antigen Tests

A

 Influenza, RSV, Adenovirus
 Access vs. Quality
– Insensitive
– Available anywhere with a lab, and some places without.

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15
Q

Viral Molecular Diagnostics

A 
 Detection of viral DNA or RNA
– PCR (many vendors)
– SDA (BD)
– TMA (Gen-probe)
 Rapidly evolving
 Remember a reverse transcriptase step (RT-PCR) is
required to detect RNA viruses
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16
Q

Molecular Applications

A

 Detection
– Rapidly becoming test of choice for many viruses
 Herpesvirus and enterovirus CNS infections.
 Respiratory viral infections.
 Caliciviruses
 HPV
 Many others
– Optimized for sensitivity.
– Use of internal controls to detect inhibition.
– Real-time methods to limit contamination and save labor.
– Automated extraction systems

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17
Q

Molecular Applications

A

 Viral load (HCV, HIV, others)
– Use of internal calibration standards to control for
extraction efficiency.
– Standardization needed.
 Viral genotyping
– HCV: viral subtype affects duration and intensity of therapy, prognosis
– HIV: drug susceptibility testing by sequencing

18
Q

Viral Serology

A 
 Detect antibody to viral antigens
 Automated, other EIA, rapid tests
 Primary diagnosis
– HIV, HCV, HBV, EBV
 Immune Status Testing
– HBV, VZV, MMR, etc.
 Issues
– Screening vs confirmation
– Cutoff values
– Western blots and RIBAs and other confirmatory strategies
19
Q

Viral Histopathology

A

Selected examples of viral cytopathic effects
in tissues, with inclusion bodies (when present)

need to type some of these in

20
Q

Adenovirus

A

 ds DNA, non-enveloped, replicates in nucleus
– >47 serotypes, six sub-genera ( A to F )
 Wide variety of syndromes
– Pneumonia in immunocompromised patients & military
recruits
– Acute gastroenteritis children (40,41)
– Pharyngitis, pharyngoconjunctival fever
– Keratoconjunctivitis
– Hemorrhagic cystitis
– Cervicitis, urethritis
– Disseminated disease

•Alveolar lining cells contain large, homogeneous, very
characteristic intranuclear inclusions: “Smudge cells”

Pearls from the inclusion body:
• Nuclear only, not cytoplasmic
• Large, deeply basophilic (blue) with NO halo
• Nucleo-cytoplasmic blurring, small amount of peripheral
cytoplasm left
• No cytomegalia
• No multinuclearity
• No smooth ground-glass appearance
• Very coarse, irregularlooking, “smudgy” material
• “SMUDGE CELLS”

21
Q

HSV 1 & 2

A

 All herpes family viruses are enveloped ds DNA
viruses.
 Three subfamilies:
– Alphaherpesviruses - HSV-1, HSV-2, VZV
– Betaherpesviruses - CMV, HHV-6, HHV-7
– Gammaherpesviruses - EBV, HHV-8
 Set up latent or persistent infection following primary
infection ( ie., “they never divorce you!!!” )
 Reactivations are more likely to take place during
periods of immunosuppression

22
Q

Varicella-zoster Virus (VZV)

A

 Chickenpox (Primary varicella); vesicular, disseminated
 Herpes zoster (Reactivation); vesicular, dermatomal
 Beware! In an immunocompromised patient, a disseminated rash may be a case of:
– Primary Varicella
– Disseminated Zoster
– Disseminated HSV

23
Q

Herpes simplex virus (HSV) & Varicella-Zoster Virus

(VZV) produce inclusions that are practically identical

A

Multinucleated giant cells with ‘molded’ nuclei

Intranuclear inclusion with marginated chromatin

24
Q

CMV (Human Cytomegalovirus)

A

Large (‘megalic’) blood-vessel lining cell (‘endothelial’) ballooning into the lumen

Intranuclear ‘Owl’s-eye’ inclusion with a halo

Abundant pink (eosinophilic) intracytoplasmic inclusions

25
Q

Paramyxoviruses

A

may produce syncytia in tissue: RSV, Parainfluenza, measles

26
Q

Rabies

A

Negri bodies: intracytoplasmic eosinophilic inclusion bodies

27
Q

Hepatitis Serology

A

Used for diagnosis, prognosis, and immune status monitoring.

Antigen is bad:
– Implies viral activity, associated with:
 Active disease (HBsAg)
 Poor prognosis (HBeAg).
 Positive antibody result is associated with resolved disease (HBsAb) or better prognosis (HBeAb).
 Markers of acute infection are core IgM (HBcAb-IgM) and surface antigen (HBsAg):
– Antigen produced before immunity
– IgM, like always, is the first antibody subtype made.
 Core antibody (HBcAb) is the test for exposure to HBV:
– Screening marker for blood
– Almost invariably present in infected patients past the acute phase
– Doesn’t reflect disease activity.
 Positive surface antibody (HBsAb) alone means immunization:
– The HBV vaccine contains surface antigen but no core antigen.
Immunized people will be HBsAb positive but HBcAb negative.

28
Q

Hepatitis C

A

Mostly blood-borne
– sexual transmission rare.
– perinatal transmission risk is <25% of infected persons
– Fulminant HCV hepatitis is very rare except in Japan.
 Chronic hepatitis C
– 50-85% of persons infected with HCV develop persistent, viremic infections
– Asymptomatic or only mildly symptomatic
– Persists for decades, with fluctuating ALT / AST levels.
 Associations
– Cryoglobulinemia
– Kidney disease (membranoproliferative glomerulonephritis)
– Other autoimmune diseases
– Lymphomas and hepatocellular carcinoma

29
Q

Hepatitis C Tests

A

Hepatitis C antibody - Anti-HCV
Becomes positive 6-8 weeks after infection. Marker of HCV infection

Hepatitis C recombinant immunoblot - HCV RIBA Used to confirm borderline HCV antibody test

Hepatitis C RNA - HCV RNA, HCV viral load, etc. Viral load (amount of virus in blood) by quantitative PCR is used to decide on and monitor therapy. For diagnosis, these are supplemental tests

Hepatitis C Genotype - Provides information on viral strains used for prognostic purposes and to guide treatment decisions. Strain types 2 and 3 are more likely to respond
to treatment than strain type 1

30
Q

HIV Tests

A 
- HIV Screening EIA 
1st gen: crude viral lysate
2nd gen: recombinant antigens
3rd gen: improved IgM detection
4th gen: antibody and antigen detection

-HIV Confirmatory Tests
Western blot
IFA

  • HIV DNA PCR
    For neonatal diagnosis
  • HIV RNA PCR
    for primary screening of blood products
    for diagnosis
    for viral load testing
  • HIV genotyping
    drug resistance monitoring by DNA sequencing; RT
    and PR mutations
31
Q

HIV Western Blot

A

Positive is:
– CDC/ASTPHLD criteria;
any two of the following bands: p24, gp41, and gp120/160

32
Q

Influenza Testing

A

 Culture
– Newer shell vial methods 24-48h
– ‘Gold standard’
 DFA
– Rapid (1-2h)
– Sensitivity from top centers approaches culture.
– Requires fluorescent scope and highly trained technical staff.
 Molecular Diagnostics
– Real-time methods can be ~1h or so.
– In some cases real-time PCR methods exceed culture in sensitivity
(probably due to viral loss in transport)
– High skill and equipment requirements
– Emerging ‘gold standard’ – overall probably better than culture
 Rapid antigen tests
– 50-70% sensitivity, despite publications to the contrary
 Lower in adults
– Rapid, simple, no equipment needed.

33
Q

Decisions Based on Rapid Flu

A 
 Positive Test
– Do not start antibacterials
– Start antivirals
– Droplet precautions
– Don’t do other tests
 Consequences of error: potentially severe
 Specificity is essential!
 Negative Test
– Consider antibacterial therapy
– Do not start antivirals
– No droplet precautions
– Further diagnostic studies
 Consequences of error: moderate
 Insensitivity can be tolerated
34
Q

It’s All About the Flu

A

 Two major considerations for influenza
testing:
–When to test?
When do you start / stop testing for flu?
–What specimens to collect?
 How do you train your clinical staff in
specimen collection?

35
Q

When to test for flu?

A

When?
 Remember – false-positives have potentially
severe consequences, e.g. non-treatment of a serious bacterial infection.
 Test during the flu season, only.
 Potential strategies:
– Seasonal: test Oct-Dec→March or so.
 Early season – retain specimen for confirmatory testing!
– Incidence-based testing – monitor regional
influenza per CDC and State systems, begin testing only when influenza reported in the area.

36
Q

Influenza Specimen Collection

A

Specimen collection is the critical step in
influenza testing

Washes are somewhat better than swabs*

 Important to get ciliated epithelial cells
 Children shed more virus than adults;
tests tend to be more sensitive in kids.

37
Q

Respiratory Viruses

A 
 Upper / Colds
– Rhinovirus
– Respiratory syncytial virus
– Adenovirus
– Parainfluenza
– Influenza A,B
– Enterovirus
– EBV
 Lower / Pneumonia
– Influenza A, B
– Adenovirus
– Respiratory syncytial virus
– Human metapneumovirus
– Parainfluenza
– Rhinovirus
– Cytomegalovirus
– Varicella-zoster
– Herpes simplex
– Hantavirus*
– SARS CoV*
38
Q

Gastroenteritis Viruses

A 
 Rotavirus
 Norovirus
 Adenovirus
 Enterovirus
 CMV (compromised patients; colitis)
39
Q

Meningitis / Encephalitis Viruses

A 
 Meningitis
– Enterovirus
– Herpes simplex virus
– Varicella-zoster
– EBV
– Mumps
– WNV
– Jamestown Canyon
– Lymphocytic choriomeningitis virus (LCMV)
 Encephalitis
– Herpes simplex virus
– Cytomegalovirus
– Varicella-zoster
– EBV
– Arbovirus (EEE, WEE, SLE,West Nile, POW, etc)
– Adenovirus
– Measles, Rubella, Mumps
– Influenza
– Enterovirus
– HIV
– BKV
– HHV-6
– Rabies
– LCMV (transplant)
40
Q

Skin

A 
 Vesicular
– HSV, VZV, Enterovirus, Vaccinia, CMV,
Adenovirus
 Papillomas
– HPV, molluscum contagiosum
 Exanthems
– Measles. Rubella, Enterovirus, Parvovirus B19, HHV-6, Dengue, West Nile, EBV, Adenovirus, CMV
41
Q

Hepatitis Viruses

A 
 Hepatitis A, B, C, D, E
 HSV, especially in newborns
 EBV
 CMV
 Adenovirus
42
Q

Other Syndromes

A

 Mononucleosis
– EBV, CMV, Adenovirus, HIV, HHV-6
 Ocular
– Enterovirus, Adenovirus, HSV, VZV, CMV,
Vaccinia, Measles
 Marrow Suppression
– EBV, MV, HHV-6, Hepatitis A, B, C, Parvovirus B19, Influenza, Adenovirus, HIV

CP Board Review - Microbiology (6 decks)

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