Download The war against infectious diseases has been won Viral Disease

Clinical
Clinical and
and Diagnostic
Diagnostic Virology
Virology
-- an
Introduction
an Introduction
The war against infectious
diseases has been won
US Surgeon General 1967
Bill Rawlinson
[email protected]
1.
Scope of clinical virology
-
2.
Scope of diagnostic virology
-
3.
Patients
populations
Principles
-
Viral
Viral Disease
Disease
Children
Adults
Special groups
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
• Oldest recorded disease (Rabies, polio)
• Modern epidemics/pandemics – HIVAIDS, HCV, SARS, Avian Influenza
• Impact on
- humans
- animals
- plants
- evolution
Viral
Viral Syndromes
Syndromes
•
•
•
•
•
•
•
•
Adenopathy and glandular fever
Arthritis
Carditis
Chronic Fatigue Syndrome
Congenital and perinatal disease
Exanthemata and skin disease
Eye disease
Gastroenteritis
Viral
Viral Syndromes
Syndromes
•
•
•
•
Haemorrhagic fevers
Hepatitis
Immunocompromised infections
Neurological disease
- encephalitis and meningitis
• Pancreatitis and diabetes
• Respiratory disease
• Sexually Transmitted Infections (STD, STI)
1
1.
Scope of clinical virology
-
2.
Children
Adults
Special groups
Scope of diagnostic virology
-
3.
Patients
populations
Principles
-
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
2
1.
Scope of clinical virology
-
2.
Scope of diagnostic virology
-
3.
Patients
populations
Principles
-
Epidemics
Epidemics
Children
Adults
Special groups
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
• Measles
– Plague of Athens (436 B.C.) described a
distemper-like epidemic with high mortality
– Epidemics - Rome, China AD165, AD251
– Considered a normal process of
development
– All adults survivors of childhood infection in
Europe
3
Epidemics
Epidemics
• Smallpox
– Earliest accounts from India, in Sanskrit
medical text, China, 1122 B.C.
– Entered Europe via Islamic North African
expansion to Spain; epidemics in Syria
(A.D.302) and Mecca (A.D. 569)
– Reintroduced to Europe via crusaders
– Disease milder limited to children (Spain in
the 1400s)
Blood
Blood supply
supply –– viral
viral infection
infection
risks
risks
• Single unit (USA)
(USA post NAT) 1 : 1.9 million (HIV)
1 : 1.6 million (HCV)
1 : 0.2 million (HBV)
• HIV and HCV infection 2 – 3 times
greater in first time donors
Blood
Blood Supply
Supply testing
testing
• Mainly viral
• Ignores persistent non-threatening
viruses
• Constantly changing
• Role of emerging viruses
Blood
Blood supply
supply –– current
current testing
testing
•
•
•
•
•
HBV
HCV
HIV 1/2
HTLV I/II
Syphilis
[Germain, 2004]
Agents
Agents of
of risk
risk –– known
known //
tested
tested
• HBsAg - Sept 1970
•
- Abbott PRISM
• HCVAb - Feb 1990
- Abbott PRISM •
• HIV 1/2 Ab - April 1985
- Abbott PRISM
• HTLV I/II Ab - Jan 1993
•
- Abbott
PRISM
HCV RNA - June 2000
- Pooled 24, TMA Chiron
- Pooled 16, April 2005
HIV 1 RNA - June 2000
- Pooled 24, TMA Chiron
- Pooled 16, April 2005
CMV - selected
Agents
Agents of
of risk
risk –– known
known // not
not
tested
tested
• CMV
• GB-C virus unknown
• HHV8
– few donors
– theoretical KS
• Prions vCJD
– few donors
– transmissible
encephalopathy
• SARS coronavirus - few
donors
• TT virus – 80-95% of
donors
• SEN-V
– 2% of donors
– types A-H
– hepatitis
• WNV
– few donors
– 3 week deferral process
– encephalitis
4
Agents
Agents of
of risk
risk –– known
known //
emerging
emerging risk
risk
• Dengue - deferral procedures for at-risk
• Prions - vCJD [Llewellyn, 2004]
- exclusion
1.
Scope of clinical virology
-
2.
Scope of diagnostic virology
-
3.
• SARS coronavirus
• WNV
• Unknown
[ Chamberland, 2001]
Children
Adults
Special groups
Patients
populations
Principles
-
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
Definitions
Definitions
A virus is a molecular genetic
parasite that uses cellular systems
for its own replication
[Villarreal, 2005]
Capsid
Protein coat surrounding viral DNA or RNA.
Made up of smaller subunits (capsomers) that
self-assemble into symmetrical helices (all of
which are enveloped) or icosaherons (with
cubical symmetry)
Envelope
Lipoprotein surrounding the nucleocapsid
Ligand
Receptor binding molecule of a virus
Light
microscope
Level of resolution is 200 nm, most viruses are
<200 nm diameter
Definitions
Definitions
Prion
Infectious protein, containing no DNA or RNA
Replication
Growth
Virion
Infectious viral particle
Viroid
Simplest virus-like organism, with infectious RNA
and no protein coat
Virus
Infectious particle consisting of DNA or RNA, ±
protein coat (capsid), ± lipid coat (envelope)
cannot be seen with a light microscope of
resolution 200 nm
Structure
Structure
• Viruses are:
– not cells
– dependent upon the cell they infect. Inside cells
they can replicate, outside cells they can be
transmitted, but cannot replicate (grow)
– contain DNA or RNA but not both
– can grow from only virus DNA or RNA inside a cell
– sometimes viruses integrate their nucleic acid into
the host cell genome
5
Viruses
Viruses
• This intimate relationship between the
virus and the cell causes several
important effects:
– Viruses are not killed by antibiotics
– Antivirals often damage the cell
– Viruses can persist in cells either
replicating (HIV) or resting – latent (HSV)
Viruses
Viruses have
have life
life
•
•
•
•
Can be killed
Can become extinct
Undergo Darwinian selection
Subject to evolutionary biology
Viruses
Viruses
• Viruses are the simplest organisms,
containing DNA or RNA, but not both.
• Prions are similar, but distinct, only
contain protein. Original concept was a
growing organism like a virus or a
human had to contain either DNA or
RNA (virus) or both (humans). Prions
are the only known exception
But
But viruses
viruses
• Have no sexual exchange process
• Species is defined by its lineage
• Species is a class that occupies a
replicating lineage and occupies an
ecological niche
6
1.
Scope of clinical virology
-
2.
Children
Adults
Special groups
Scope of diagnostic virology
-
3.
Patients
populations
Principles
-
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
1cm
HSV-2
HSV-2 EM
EM
eye
1mm
plant cell
10µm
animal cell
100nm
10nm
(Linda Stannard, University of Cape Town, S.A.)
Classification
Classification
• Standard levels
– Order
– Family
– Subfamily
– Genus
– Species
thickness of a 5 cent coin
100µm
1µm
light
microscope
electron
microscope
smartie
1nm
0.1nm
Log scale
bacterium
virus
protein
small molecule
atom
Classification
Classification
• Classification is based on:
~virales
~viridae
~virinae
~virus
– Genomic makeup e.g: Caliciviruses
– Virion structure – EM appearance e.g: HV
– Replication strategy
– Virion antigenicity e.g: adenoviruses,
serological distinction MVE / JE / WNV
– Virion chemical characteristics, stability
– Diseases caused in the host e.g: hepatitis
7
Classification
Classification
• Recognition of virus (ICTV) in 2005 →
1550 species
• Information available on the ICTV
database at
www.ncbi.nlm.nih.gov/ICTVdb
Viral
Viral structure
structure
• Can only be two types
– helical (all of these are enveloped in animal
viruses), which looks like a tube
– icosahedral (cubical) which is round
1.
Scope of clinical virology
-
2.
Children
Adults
Special groups
Scope of diagnostic virology
-
3.
Patients
populations
Principles
-
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
Virion
Virion Architecture
Architecture
Architecture of virions regardless of host
is based on two simple themes:
Sphere – normally in the form icosahedron (cubical)
Best way of producing a shell of equivalently bonded
identical structures
Minimum free energy state
Strong structure that can enclose a maximal volume
Helix – cylindrical shape (spiral staircase)
Viruses
Viruses as
as aa molecule
molecule
• Host viruses
– 10 – 20 genes
– Genomes 5,000 – 25,000 bp
• ICTVdb
– 3,600 species
– 30,000 strains + subtypes
8
Biological
Biological characteristics
characteristics of
of acute
acute
and
persistent
virus
life
strategies
and persistent virus life strategies
• Persistent virus life strategy
–
–
–
–
–
–
Persistent in individual host
Seldom causes acute disease; often inapparent
Genetically stable
Virus is highly species specific
Virus often shows coevolution with host
Transmission is often from parent to offspring
(vertical) or through sexual contact
– Less dependent on host population structure
– Often the source of emerging acute disease in
new host species
1.
Viruses
Viruses as
as aa molecule
molecule
•
•
•
•
Biological
Biological characteristics
characteristics of
of acute
acute
and
and persistent
persistent virus
virus life
life strategies
strategies
Scope of clinical virology
-
2.
Children
Adults
Special groups
Scope of diagnostic virology
-
3.
Patients
populations
• Acute virus life strategy
–
–
–
–
–
–
–
–
Principles
-
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
Examples
Examples of
of virus
virus structure
structure
ssRNA – most diverse Noro, HCV, HIV
dsDNA – Adeno, CMV, HSV, Variola
dsRNA – Rota
ssDNA – least diverse PVB19
1.
No persistence in individual host
Often disease associated
High mutation rates (RNA viruses)
Virus replicates in more than one species
Virus does not show coevolution with host
Transmission is horizontal
Highly dependent on host population structure
Seldom evolves to persistence
Scope of clinical virology
-
2.
Children
Adults
Special groups
Scope of diagnostic virology
-
3.
Patients
populations
Principles
-
Definitions
Classification
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
9
Types
Types of
of diagnostic
diagnostic test
test
• Open
- Cytology
- EM
- Culture
• Closed -
NAT
LA
Solid phase ELISA
Specific serology
Diagnostic
Diagnostic Methods
Methods
• Serology - restrospective
• Ag
–
–
–
–
Protein based
IFA (Respiratory)
WB (HIV)
Protein function (HIV-RT)
• Culture – some viruses non-cultivable
• Molecular
– Virion nucleic acid
– HCV RNA, HCV RNA
– CMV DNA
• Emerging
Serology
Serology
•
•
•
•
ELISA, IFA, CFT
Total (EV, HAV, HCV, HW, Influenza)
Igm (Adeno, HSV)
IgG (CMV, Mumps, Measles, PVB19
Rubella)
• Complex (HBV, EBV)
• No use (Rota, Noro, Variola)
Molecular
Molecular testing
testing
• Rapid, sensitive, costly (initially)
• Amplified
– Signal (PCR, LCR, NASBA, TMA)
– Signal (bDNA)
• Non amplified
– Probe based (ISH, Hybrid Capture)
NAT
NAT target
target
• RNA/DNA
• Qualitative/Quantitative
• Detection/Infection
• NAT Issues
10
Comparison of Amplification
Methods
NAT
NAT –– interpretation
interpretation issues
issues
•
•
•
•
Virus,
bacterium
or cell
Infection – disease
RNA – DNA
Replication competent – genomic
Reference standards
Target RNA or DNA
TMA
– Culture
– Antibody testing
– None
PCR
Add
primers &
enzymes
Copies
(RNA)
Copies
(DNA)
Multiplex
Multiplex PCR
PCR
280
240
208
131
104
CMV
VZV
T.gondii
1
detection
probe per
amplified
copy
Ag
Ag testing
testing
396
350
222
179
126
HSV
Add
probes
and
branched
DNA
Multiple
detection
probes
per target
1
detection
probe per
copy
• Tissue source
bDNA
Add
primers &
enzymes
•
•
•
•
– many host cells in specimen
– operator
PVB
Tests
Tests –– emerging
emerging techniques
techniques
Existing technique (Respiratory)
Initial testing (p24)
Only available (Prion)
Dependent upon
1.
Scope of clinical virology
-
2.
- optical bead arrays
- protein nanoarrays
Scope of diagnostic virology
-
• New arrays - microarray
3.
Patients
populations
Principles
-
• Redevelopment of existing methods
Children
Adults
Special groups
Definitions
Classifications
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
11
Herpes
Herpes Simplex
Simplex Encephalitis
Encephalitis
•
•
•
•
•
Most common cause of encephalitis
1-4/1,000,000 per annum
50% have long term sequelae
80% untreated mortality
Associated with reactivation or primary
infection
HSE
HSE -- MRI
MRI
HSE
HSE
12
HSV
HSV PCR
PCR detection
detection after
after
treatment
treatment
HSE
HSE –– HSV
HSV PCR
PCR negative
negative
• Can be PCR negative early in course
(<3 days)
• Repeat CSF analysis after 3 days of
treatment
» e.g. Tiege XD et. al. Clin Infect Dis 2003; 36:
1335
18/18
100
90
80
70
60
50
40
30
20
10
0
49/50
8/17
4/19
Percent
Positive
< Day 0
D 0-7
D 8-14
> D 15
No correlation
between
persistence and
outcome
• Lakeman FD et. al. J Infect Dis 1995; 171: 857
HSE
HSE -- EEG
EEG
• Less sensitive than MRI
• Typically non-specific in first 5 days
• Lateralising periodic sharp and slow
wave pattern
Encephalitis
Encephalitis other
other tests
tests
• Talk to laboratory
• Japanese encephalitis
– CSF IgM
– Nearly all positive by day 3 of illness
• Rabies
– RT-PCR on saliva and CSF
» Wacharapluesadee S, Lancet 2001; 358:892-893.
• Brain biopsy
– Only if non-invasive tests negative
1.
Some
Some Emerging
Emerging Issues
Issues
Scope of clinical virology
-
2.
Children
Adults
Special groups
• New respiratory virus – SARS
Scope of diagnostic virology
-
3.
Patients
populations
• Transfusion – HCV
Principles
-
– hMPV
– Associations with chronic conditions
Definitions
Classifications
Molecular and viral basics
Pathogenesis of disease
4.
Diagnosis
5.
Continuing Changes
-
methods
scenarios
– Non A non B non C
– New viruses (GBV, TTV, Sen V)
• Old viruses re-emerging
– Smallpox
• Zoonoses
–
–
–
–
SARS
Rabies
Arenaviruses
Hantaviruses
13
Leadership
Leadership
• The Duke of Wellington after his first meeting
as prime minister, with his ministers:
• “I gave them their orders, but they insisted on
discussing them”
Future
Future projections
projections -- Costs
Costs
• Laboratory costs will increase as residual
risks decrease
• HIV
- serologic testing 1: 1.5 million
- NAT testing 1: 2 million
$10 million per infection prevented (US)
$1.2 million per QALY (Europe)
$15 million per infection prevented (Aust)
• Testing single units not pooled
[Goodman, 2004]
Prion
Prion diseases
diseases
•
•
•
•
•
•
•
•
Transmissible spongiform encephalopathies
Human and animal forms
Rare
Aberrant protein folding
No treatment
Invariably fatal
Cause neurodegeneration
Iatrogenic transmission concerns
Variant
Variant CJD
CJD
• Bovine Spongiform Encephalopathy
• Younger
• 60% have psychiatric symptoms initially –
anxiety, insomnia, withdrawal
• 50% have 14-3-3 protein in CSF
• Tau protein in CSF
• MRI – high T2 signal in posterior thalamus
• EEG not useful
14