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Influenza C in Alberta
TARRANT Symposium 2012
Kanti Pabbaraju
Lab Scientist
ProvLab
[email protected]
Matsuzaki et al., JID, 2006
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Tested 84946 patients from 1990 to 2004
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170 (0.22%) were positive for FluC
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Excluded all co-infections
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Retrospective chart review
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Studied symptoms and compared hospitalized
Vs non-hospitalized patients
Clinical features associated with influenza C virus infection.
Matsuzaki Y et al. J Infect Dis. 2006;193:1229-1235
© 2006 by the Infectious Diseases Society of America
Clinical diagnoses in influenza C virus–infected children.
Matsuzaki Y et al. J Infect Dis. 2006;193:1229-1235
© 2006 by the Infectious Diseases Society of America
Comparison of clinical features of type C and A influenza virus infections at Katsushima
Pediatric Clinic during January–March 2002.
Matsuzaki Y et al. J Infect Dis. 2006;193:1229-1235
© 2006 by the Infectious Diseases Society of America
Results
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92% were less than 6 years old
Fever, cough and rhinorrhea were the most
common symptoms
29 were hospitalized and 21 of these had LRT
such as pneumonia, bronchitis and brochiolitis
Rate of hospitalization was significantly higher in
<2 year olds
In addition
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A case of acute encephalopathy associated with influenza C has
been reported (Takayanagi, 2009).
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Documented as the etiological cause of several outbreaks in
schools and the community (Ramos 2008, Matsuzaki 2007,
Matsuzaki 2002, GreenBaum 1998, Katagiri 1987, Katagiri 1983)
Thus overall burden of Influenza C infections should not be underestimated
Why is FluC under-diagnosed
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Few reports describing its clinical features
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Difficulty in isolating it (No suitable cell lines)
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Amniotic inoculation of embryonated hen's eggs has been
employed to isolate the virus from clinical specimens.
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Attributable to mild pathogenecity
Important epidemiological features
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Antigenically and genetically different strains co-circulate
in a community
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Genetic re-assorting occurs frequently among strains
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Newly emergent re-assortant viruses become
predominant
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Humans with antibodies to FluC can be repeatedly
infected
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Pigs and dogs have been reported to have antibodies
against Influenza C, thus it can cause zoonooses
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Re-assortants with pig and human FluC genes reported
Design of RT-PCR assay
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Assay Design
 Real-time PCR assay using labelled probes on the Taqman platform
 Target the conserved matrix gene
Strong pos
Weak pos
Neg
Sensitivity, dynamic range,
reproducibility, linearity
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Limit-of-detection was determined using quantified Influenza C RNA
prepared in-vitro
Copy number for in-vitro RNA
Average Ct
SD
%CV
4.51E+08
11.52
0.08
0.68
4.51E+07
14.89
0.07
0.44
4.51E+06
18.24
0.05
0.25
4.51E+05
21.67
0.04
0.19
4.51E+04
25.07
0.07
0.28
4.51E+03
28.63
0.07
0.26
4.51E+02
31.91
0.10
0.32
4.51E+01
35.39
0.44
1.24
4.51E+00
39.18
0.89
2.28
Sensitivity, dynamic range,
reproducibility, linearity
Detection of a range of FluC viral loads - replicates
45
R2 = 0.9998
40
y = -3.4407x + 41.18
R2 = 0.9998
35
30
25
20
15
10
5
0
0.00E+00
1.00E+00
2.00E+00
3.00E+00
4.00E+00
5.00E+00
6.00E+00
Good linearity over a large dynamic range
7.00E+00
8.00E+00
9.00E+00
1.00E+01
Specificity
Test high copy number samples of common respiratory pathogens
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different strains of influenza virus A and B,
parainfluenza virus 1, 2, 3, 4A and 4B,
RSV A and B,
human coronaviruses 229E, NL63, HKU1 and OC43
human bocavirus,
coxsackievirus A16 and B6, echovirus 2,
human metapneumovirus,
rhinovirus type 1B,
adenovirus type 4,
Legionella pneumophila,
Mycoplasma pneumoniae,
Bordetella bronchiseptica,
B. holmseii,
B. parapertussis,
B. pertussis,
Hemophilus influenzae,
Neisseria meningitidis
Streptococcus pneumoniae.
Population screened
From Sept 1, 2010 to April 30, 2011
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Children less than 10 years during hospital visits (n=427)
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Respiratory outbreaks (n=47)
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Randomly selected to include 55 samples per month
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Multiple samples from the same patient were excluded
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All specimens had tested negative for influenza A and B, RSV, human
metapneumovirus, parainfluenza types 1 to 4, coronavirus 229E, OC43, NL65 and
HKUI, adenovirus, entero/rhinovirus
Positive sample types
Specimen type
Total tested
Total positive % positive in specimen type
Nasopharyngeal swab
313
7
2.24
Auger suction fluid
67
3
4.48
Nasopharyngeal fluid
45
1
2.22
Throat swab
24
0
0.00
Others
25
0
0.00
Nasopharyngeal swab
Nasopharyngeal fluid
Others
n=45
Positive=1
n=24
Positive=0
Auger suction fluid
Throat swab
n=25
Positive=0
n=314
Positive=7
n=67
Positive=3
Age distribution
300
# of Samples tested
2.76
3.00
% positve
250
254
2.50
2.41
2.15
200
2.00
150
1.50
100
1.00
93
83
44
50
2
7
2
0.50
0
0
0.00
<1
1 to 5
Age in years
5 to 10
10 to 100
Percent positive
Number of samples
# of Positives detected
Seasonality
80
7.00
Total tested
5.66
6.00
%positive
60
5.00
50
3.77
4.00
40
3.00
30
2.00
1.56
20
1.79
1.00
10
1
1
3
4
2
0
0.00
Sep 10
Oct 10
Nov 10
Dec 10
Jan 11
Month-Year
Feb 11
Mar 11
Apr 11
Percent positive
Total positives
70
Number of samples
6.25
Monthly isolation of influenza C virus between December 1990 and November 2004.
Matsuzaki Y et al. J Infect Dis. 2006;193:1229-1235
© 2006 by the Infectious Diseases Society of America
Phylogenetic tree for HE gene
Aichi_81
Kansas_79 C/Aichi/1/81
Georgia_69
Johannesburg_66
Aichi_99
93% identity
C11VC3502
SaoPaulo82
C/Sao Paulo/378/82
Yamagata93
42 isolates (1947-1993)
revealed six lineages
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co-circulation was detected
Paris_67
Taylor_47
C/Taylor/1233/47
M11VC4753
M11VC10161
C11VC2921
C/ Kanagawa/1/76
Catalonia2009
M11VC4941
>98% identity
Singapore_2006
C11VC3087
Yamagata_2004
Kanagawa_76
Fukuoka_2004
Yamagata_98
Miyagi_92
England_83
Pig_Beijing_81
Yamagata_88 C/Yamagata/26/81
Yamagata_81
NewJersey_76
Kyoto_79
Sapporo_71
Greece_79
Mississipi_80
C/Mississippi/80
5.8
4
2
Nucleotide Substitutions (x100)
0
Co-circulation of different lineages
Leads to re-assortment and epidemics of the new strain
Phylogenetic tree for M gene
Aichi_81
Kansas_79
Johannesburg_66
Mississippi_80
Greece_79
Taylor_47
Sao
Yamagata_93
Kanagawa_76
Kyoto_79
C/Yamagata/26/81NewJersey_76
Yamagata_81
related lineage
Yamagata_88
C/Aichi/1/81- or
C/Mississippi/80related lineage
Pig_Beijing_81
Aichi_99
England_83
M11VC002193
M11VC004941
M11VC010161
C11VC002921
C11VC004753
M10VC021100
C11VC003087
>98% identity
Miyagi_92
2.6
C11VC002616
C11VC003502
C11VC004406
C/Aomori/74
2
0
Nucleotide Substitutions (x100)
Conclusions
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Sensitive and specific for the detection of Influenza C
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Good reproducibility
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Is linear over a large dynamic range
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Will facilitate for testing of influenza C viruses in
respiratory samples in a high throughput fashion
Future work
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screening of patient samples including children and
adults
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Influenza C in outbreaks of unknown etiology
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Studying the epidemiology
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Are FluC infections cyclical or endemic
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Role of FluC in hemopoeitic dysfunctions
Better strain characterization of Influenza C isolates in
the community
Acknowledgements
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Dr. Kevin Fonseca
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Dr. Raymond Tellier
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Sallene Wong
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Anita Wong
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Vinod Khurana and the MOD group