influenza c in alberta tarrant symposium 2012 kanti pabbaraju lab scientist provlab...
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Matsuzaki et al., JID, 2006 Tested 84946 patients from 1990 to 2004
170 (0.22%) were positive for FluC
Excluded all co-infections
Retrospective chart review
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
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 A case of acute encephalopathy associated with influenza C has
been reported (Takayanagi, 2009).
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
Few reports describing its clinical features
Difficulty in isolating it (No suitable cell lines)
Amniotic inoculation of embryonated hen's eggs has been
employed to isolate the virus from clinical specimens.
Attributable to mild pathogenecity
Important epidemiological features Antigenically and genetically different strains co-circulate in a
community
Genetic re-assorting occurs frequently among strains
Newly emergent re-assortant viruses become predominant
Humans with antibodies to FluC can be repeatedly infected
Pigs and dogs have been reported to have antibodies
against Influenza C, thus it can cause zoonooses
Re-assortants with pig and human FluC genes reported
Design of RT-PCR assay 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 Limit-of-detection was determined using quantified Influenza C RNA
prepared in-vitro
Copy number for in-vitro RNAAverage
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
y = -3.4407x + 41.18
R2 = 0.9998
0
5
10
15
20
25
30
35
40
45
0.00E+00 1.00E+00 2.00E+00 3.00E+00 4.00E+00 5.00E+00 6.00E+00 7.00E+00 8.00E+00 9.00E+00 1.00E+01
R2 = 0.9998
Detection of a range of FluC viral loads - replicates
Good linearity over a large dynamic range
Specificity
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.
Test high copy number samples of common respiratory pathogens
Population screened
From Sept 1, 2010 to April 30, 2011
Children less than 10 years during hospital visits (n=427)
Respiratory outbreaks (n=47)
Randomly selected to include 55 samples per month
Multiple samples from the same patient were excluded
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 Auger suction fluidNasopharyngeal fluid Throat swabOthers
n=24Positive=0n=45
Positive=1
n=67Positive=3
n=314Positive=7
n=25Positive=0
Age distribution
44
2 7 2 0
254
9383
2.15
2.41
2.76
0
50
100
150
200
250
300
<1 1 to 5 5 to 10 10 to 100
0.00
0.50
1.00
1.50
2.00
2.50
3.00# of Samples tested
# of Positives detected
% positve
Age in years
Per
cent
pos
itive
Num
ber
of s
ampl
es
Seasonality
1 13 4
2
3.77
1.561.79
5.66
6.25
0
10
20
30
40
50
60
70
80
Sep 10 Oct 10 Nov 10 Dec 10 Jan 11 Feb 11 Mar 11 Apr 11
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Total tested
Total positives
%positive
Month-Year
Num
ber
of s
ampl
es
Per
cent
pos
itive
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
Nucleotide Substitutions (x100)0
5.8
24
Aichi_81Kansas_79
Georgia_69Johannesburg_66
Aichi_99C11VC3502
SaoPaulo82Yamagata93
Paris_67Taylor_47
M11VC4753M11VC10161C11VC2921
Catalonia2009M11VC4941
Singapore_2006C11VC3087
Yamagata_2004Kanagawa_76
Fukuoka_2004Yamagata_98
Miyagi_92England_83
Pig_Beijing_81Yamagata_88
Yamagata_81NewJersey_76
Kyoto_79Sapporo_71
Greece_79Mississipi_80
C/Aichi/1/81
C/Yamagata/26/81
C/Taylor/1233/47 C/Sao Paulo/378/82
C/ Kanagawa/1/76
C/Mississippi/80
42 isolates (1947-1993)
revealed six lineages co-circulation was detected
>98% identity
93% identity
Co-circulation of different lineages
Leads to re-assortment and epidemics of the new strain
Phylogenetic tree for M gene
Nucleotide Substitutions (x100)0
2.6
2
Aichi_81Kansas_79
Johannesburg_66Mississippi_80
Greece_79Taylor_47
SaoYamagata_93
Kanagawa_76Kyoto_79NewJersey_76
Yamagata_81Yamagata_88
Pig_Beijing_81Aichi_99
England_83M11VC002193M11VC004941M11VC010161
C11VC002921C11VC004753M10VC021100
C11VC003087Miyagi_92
C11VC002616C11VC003502
C11VC004406
C/Yamagata/26/81-related
lineage
C/Aomori/74
C/Aichi/1/81- or C/Mississippi/80-
related lineage
>98% identity
Conclusions
Sensitive and specific for the detection of Influenza C
Good reproducibility
Is linear over a large dynamic range
Will facilitate for testing of influenza C viruses in
respiratory samples in a high throughput fashion
Future work screening of patient samples including children and
adults
Influenza C in outbreaks of unknown etiology
Studying the epidemiology Are FluC infections cyclical or endemic
Role of FluC in hemopoeitic dysfunctions
Better strain characterization of Influenza C isolates in
the community
Acknowledgements
Dr. Kevin Fonseca
Dr. Raymond Tellier
Sallene Wong
Anita Wong
Vinod Khurana and the MOD group