Transcript Molecular diagnosis of respiratory viruses and its impact

Molecular diagnosis of
respiratory viruses and its
impact on clinical
management
Prof G Kudesia
Sheffield Teaching Hospitals
NHS Trust
Cell Culture
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Widely used
Result in 7-14 days
or longer
Adenovirus CPE in RMK
Un-infected RMK
Live cells required
Cytopathic effect
needs to be
confirmed by
specific tests .
Technical expertise
Time delay
Catch ‘all’
Antigen detection by
Immunofluorescence
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Rapid
Relatively
insensitive
Not suitable for all
speciemn types
Subjective
Serology
Technically
demanding
Insensitive
Acute and
convalescent
serum sample
Respiratory Viruses: Diagnosis Pre 1990’s.
Advantages
Tissue
Culture
Serology
Antigen
detection
“Open” Technique
Sensitive
Further
characterisation
Evidence of active
infection

Detects current and
past infection
(immunity)
Important for
fastidious viruses
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Rapid
Detects non-viable
virus
Can test large
numbers of samples
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Disadvantage
s
Not applicable to
all
Time consuming
viable virus only
Contamination/to
xins
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Prolonged testing
time
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Not applicable to
all
interference
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Polymerase Chain Reaction
(PCR)-Xeroxing DNA!
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Kary Mullis
Won the NobelPrize
in 1993 for
describing the
methodology in
1985 to replicate
DNA in a test tube.
PCR
Impact of PCR testing on
respiratory virus investigations
Comparison of cell culture with PCR for Influenza
A and B and RSV-200 specimen tested winter
2006/07-Sheffield
Virus
RSV
PCR
38
Positive
Flu A
Rhino HMPV
Total
29
32
110
11
Cell
Culture
Positive
12
17
1
0
(32%) (59%) (3%)
Total
38
29
32
11
30
(27%)
110
Respiratory PCR from Children –winter
07/08(haematology/oncology)
50
45
Rhinovirus
'Flu A
'Flu B
RSV
HMPV
Paraflu1,2,3
Adeno
Rhino+HMPV
Dual
40
35
30
25
20
15
10
5
0
%Positive
Advantages of PCR over traditional methods-R Gunson, Glasgow
Positive
Flu A
Rhino
RSV
Total
Culture
16
2
2
20
DIF
14
n/a
6
20
Total
Isolation
20
2
8
30
PCR
41
14
15
70
Sensitivity
(Iso Vs PCR)
49%
14%
53%
43%
Improved detection rate
Flu A
Flu B
RSV
PF1
PF2
PF3
PF4
hMPV
Coronavirus
Rhino
Adenovirus
45.00
40.00
Detection rate (%)
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
1996-97
1997-98
1998-99
1999-2000
2000-01
2001-02
Respiratory season
2002-03
2003-04
2004-05
2005-06
Improvements in TRT
Accumulative percentage
100
1996-97
1997-98
75
1998-99
1999-00
2000-01
50
2001-02
2002-03
2003-04
25
2004-05
2005-06
0
0-1
2-3
4-5
6-8
Days
9-10
11-14
>14
Clinical impact
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Influenza
Treatment
 Prophylaxis
 Outbreak Management
 Control of infection
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Immunocompromised
Treatment
 Control of infection
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Treatment/prophylaxis for
influenza-start within 48 hours
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Oseltamivir
• Treat- 75 mg twice a day x 5 days
• Prophylaxis- 75mg once a day x10 days
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Speed for laboratory confirmation of essence
PCR testing was invaluable in the late
influenza B activity this winter- both for
outbreak and individual patient management
Flu outbreak- SVC west Scotland
Occurred out with flu season
Hospital X
Flu virus sequenced
Index case
Patient A – flu positive
Doctor A – flu
positive
Doctor B – flu
positive
Phylogenetic tree created
Nurse – flu
positive
Patient B – flu
positive
Patient C – flu
positive
Shown to be H3 Wisconsin
Sequence flu strain
originated from Patient A –
the index case
Flu outbreak
Hospital Y
Flu virus sequenced
Patient D – flu
positive
Doctor C – flu
positive
Phylogenetic tree created
Patient E – flu
positive
Was there a connection with hospital X?
Hospital X
No connection between
Hospital X and Y
Tree may have looked like
this
Hospital Y
BUT THERE WAS A CONNECTION
Showed both flu outbreaks
were connected
All were H3 Wisconsin
What was the connection?
Hospital Y
Hospital X
Patient D – flu
positive
Index case
Patient A – flu
positive
Doctor A – flu
positive
Doctor B – flu
positive
Doctor C – flu
positive
Nurse – flu
positive
Patient E – flu
positive
Patient B – flu
positive
Patient C – flu
positive
Patient C from hospital X
was transferred to hospital Y
Molecular epidemiology for
outbreak sequencing
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Implications
• Shows connections between
patients/staff
• Raises infection control issues
Patient transferred while ill
 Why were staff infected
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• Re-evaluate hospital procedures
 E.g. masks, gowns, gloves, hand washing
A case of Respiratory infection in
BMT-Sheffield
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37 year old male post BMT
Presented with GVHD in December 07
Third week of march 08- respiratory symptoms- ?
Infection, ? Respiratory GVHD
Respiratory and PCP PCRs- HMPV PCR positive
25/3, 7/4
Not treated initially but subsequently treated with
I/V and nebulised Ribavirin due to deterioration
in respiratory symptoms.
Died 14/4
Post-mortem histology of lung
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Sections from both the lungs show fibrin
and macrophages in the alveolar spaces
along with focal squamous metaplasia.
There are scattered large bizzare cells
with basophilic inclusions in the
cytoplasm. The features are those of an
organizing pneumonia with virocytopathic
effect suggesting of viral aetiology.
Human Metapneumovirus
Discovered in 2000.
 Paramyxoviridae
 Negative sense, Single stranded RNA
 Two genotypes A and B
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Clinical Problems
Upper respiratory infection
 Lower respiratory infection
 Non-specific symptoms
 Fatalities reported in BMT patients
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Objective
To determine the incidence
st September 2005 to 31 May 2006
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Methods
Data collection-retrospectively
 Descriptive methods
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Results
Specimen type
NO of specimens
NPA
205
BAL
73
ETS
22
Others
48
Total
348
The incidence of Respiratory
Pathogens
Total
Aden 13
MPV 11
FluA 11
FluB 17
Para 8
RSV 25
Meas 1
Total 86
(25%)
1
2
3
4
5
6
7
8
9
10
11
age
ward Cond Clinic Spec Outcome
ition al
<1
<1
2
3
5
5
10
38
39
54
68
PICU
PICU
PICU
M3
M3
M3
M3
E2
E2
P3
ITU
Bron
Bron
Bron
ALL
ALL
Onco
ALL
HIV
URTI
Nasal
Coryza
Dysp
Atypi
BMT
Coryza
NPA
NPA
NPA
NPA
NPA
NPA
NPA
NPA
NPA
NPA
NPA
Discharged
Discharged
Discharged
Finding
HMPV -4th commonest respiratory
pathogen
 Affected all age groups
 Detected in patients with both upper
and lower respiratory tract infections
 Some patients discharged before
results were available
 Further studies for clinical
significance
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New viruses- human Bocavirus
(HBoV)
Identified in 2005
 DNA virus belonging to family
Parvoviridae
 Found in respiratory secretions from
children with and with out respiratory
symptoms
 Exact role in respiratory infections to
be still worked out
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How feasible is it to introduce PCR in
routine diagnosis
Multiplex Real Time PCR
Multiplex 1
Influenza A
Influenza B
Influenza C
Matrix
NS
Matrix
Multiplex 2
HMPV A
HMPV B
Parainfluenza 1
Fusion
Fusion
HN
Multiplex 3
Parainfluenza 2
Parainfluenza 3
Parainfluenza 4
HN
HN
Fusion
Multiplex 4
HuCoV 229E
HuCoVOC43
HuCoV NL63
Nucleocapsid
Nucleocapsid
1a gene
Multiplex 5
RSV A
RSV B
Rhino
NP
NP
5-UTR
Small numbers of tests/More pathogens detected
Year
Format
No of tests
2000-01
2001-02
Tests performed (cumulative)
influenza A, B; RSV; adenovirus; picornavirus
Gel based nested
4
2002-03
influenza A, B; RSV; adenovirus; picornavirus
influenza A, B; RSV; adenovirus; picornavirus;
2003-04
5
influenza A, B; RSV; adenovirus; Rhinovirus; PF1, 2, 3
2004-05
5
influenza A, B; RSV A + B; adenovirus; rhinovirus;
PF1, 2, 3; coronavirus NL63, 229e, OC43; HuMPV
6
influenza A, B, C; RSV A + B; adenovirus; rhinovirus;
PF1, 2, 3, 4; coronavirus NL63, 229e, OC43;
HuMPV A + B
2005-06
2007-08
Real time PCR
5
influenza A, B, C; RSV A + B; adenovirus; rhinovirus;
PF1, 2, 3, 4; coronavirus NL63, 229e, OC43;
HuMPV A + B; M pneumoniae
Advantages of PCR
The utilisation of PCR conferred many advantages:
– Highly sensitive/specific
•
•
•
•
Applicable to RNA or DNA viruses
Rapid (turn around time of 24-48 hours)
Can detect multiple viruses
Products can be sequenced for
epidemiological/resistance study.
• Improved patient management and disease
surveillance
But……………..:
– Post amplification processing
– Contamination
– Prolonged testing time
– Non-automated
– Expensive to implement/expertise needed
– Qualitative (difficult to quantify)
Submitted by R Gunson
June 2003
Real time PCR:
Unlike conventional PCR:
–
–
Amplicon is visualised as the amplification progresses.
Exponential rather than endpoint analysis
–
Closed system
No
post-amplification processing
Rapid
Reduced
contamination
Automation/high
throughput/Cost effective
–
Increased sensitivity/specificity
–
More tests /less reagents/standardised cycling conditions
Disadvantages of real time PCR:
– Risk of false negative reactions (due to miss-matches).
– Number of amplicons detected is limited by the number of fluorophores.
– Expensive to implement
Submitted by R Gunson
June 2003
Examples of the benefits of real time PCR assays
in viral respiratory infection
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Gueudin et al:
• Developed a real time PCR to detect, subgroup, and quantitate RSV A and
B
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RSV A and B to be responsible to differing disease severities
Found higher viral loads in more severe infections
Elden et al:
• Developed a real time PCR for simultaneous detection of influenza A and B.
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Rapid diagnosis allowed timely therapeutic and infection control
intervention
Quantitation could be used to examine the effects of antiviral
therapy
Mackay et al:
• Developed a sensitive real time PCR for Human metapneumovirus
 Most sensitive assay currently available
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Puhakka et al:
• Examined the effect of zanamivir on the viral load of influenza
June 2003
Submitted by R Gunson
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Viral loads were reduced significantly
Summary
PCR for respiratory viruses are
sensitive and specific
 Positivity rate of 50% or greater
 Cell culture sensitivity 30-50%
compared to PCR (for viruses that
can be cultured)
 Detection rate of PCR improved
further as many viruses not
culturable .
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Summary- continued
Several viruses can be tested for at
the same time by multiplex PCR
 In-house PCR cost effective
compared to cell culture
 PCR effective epidemiological tool in
investigation of outbreaks
 Rapid and sensitive assay aids in
clinical management of respiratory
infections.
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