clinically validated - Longhorn Vaccines and Diagnostics

Report
Influenza Transmission Among Pediatric
Patients and Family Contacts
1747 Citadel Plaza • Suite 206 • San Antonio, TX 78209 •(210) 826-0910
[email protected]
Influenza Transmission Among Pediatric Patients and
Family Contacts
Background
Influenza virus: a highly evolving pathogenIn the US:
•200K hospitalizations, 35,000 deaths per yr.
•72 pediatric deaths in 2007/08
•Increased drug resistance (Tamiflu and adamanatane)
•Worldwide (as of July 6th):
•436 H5N1 bird flu cases, 262 deaths (61% fatality)
•94K H1 swine cases, 429 deaths (0.45% fatality)
Influenza Transmission Among Pediatric Patients and
Family Contacts
Background
Most clinical diagnostic laboratories have transitioned from
culture into molecular-based diagnostics such as PCR
Prime PCR System™ :
1. PrimeStore, a specimen collection kit, and
2. PrimeMix, a real-time detection mixture that brings
speed, safety, accuracy and flexibility to influenza
detection
C O L L E C T • K I L L • S T A B I L I Z E • P R E S E R V E
D I S P E N S E • A D D
N U C L E I C S • A N A L Y Z E
Influenza Transmission Among Pediatric Patients and
Family Contacts
Specific Aims
This prospective (2007-08) family study compared the sensitivities of traditional
culture, RAPID immunoassay (Remel X/pect) and PrimeMix real-time RT-PCR
(rRT-PCR) System for detecting influenza virus from: 1) symptomatic pediatric
patients and 2) asymptomatic or mildly infected family members.
Genetic analysis of influenza genes encoding hemagglutinin (HA), neuraminidase
(NA), and matrix surface (MA) proteins were performed using select nasal wash
specimens preserved in PrimeStore™ to evaluate vaccine effectiveness and drug
sensitivity within viral strains.
We characterized seven influenza A virus HA and MA genes from index patients and
infected family contacts. Specifically, we were interested in a genetic characterization
to determine the extent of mutation between and within confirmed family
transmissions.
Influenza Transmission Among Pediatric Patients and
Family Contacts
Experimental Design
100 pediatric patients meeting clinical case criteria* for influenza infection and 122
family contacts were enrolled in the study at Wilford Hall Medical Center’s Pediatric
Clinic (Lackland AFB, TX).
Nasal wash specimens were obtained and subjected to commercial antigen testing
(Remel), traditional culture and type/subtype-specific real-time RT-PCR (rRT-PCR)
analysis. Nasal washings subjected to rRT-PCR were placed (1:3) in PrimeStoreTM
Solution and extracted using the Ambion RNAqueous Micro Kit.
Real-time RT-PCR was performed using PrimeMixTM Solution. Genetic
characterization of selected clinical samples preserved in PrimeStore™ was
performed using standard RT-PCR and nucleotide sequencing.
Influenza Transmission Among Pediatric Patients and
Family Contacts
Experimental Design
Criteria for Patient Enrollment
 Pediatric subjects(<18 yrs)
 Fever ≥ 100.4 ˚F
 Cough and/or rhinorrhea
Study Demographics
 100 patients,122 family contacts
 222 subjects from 89 families
Criteria for Family Contact Enrollment
 Family household contacts (adult or child)
 Symptomatic or asymptomatic
 Accompanied patient to appointment
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results
Influenza was detected
by RT-PCR in 66/222
(31%) of enrolled
subjects, including 47
Influenza A (45 H3N2
and 2 H1N1), and 19
Influenza B
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Sensitivity and Specificity
Detection Summary by Method (N=222)
Type
Subtype
FluA
FluB
H3
H1 (human)
Rapid Antigen
33
12
NA
NA
Culture
39*
17
NA
NA
PrimeMix RT-PCR
47
19
44
2
*One reported culture-positive was not detected by PrimeMix
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Sensitivity and Specificity
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Sensitivity and Specificity
There were 11 subjects (9 Flu A and 2 Flu B)
that were Longhorn PrimeMix ™ positive
but culture negative; 3 were infected family
contacts (transmissions), and 2 of these 3
were asymptomatic at the time of testing.
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Vaccine Relatedness
Influenza A strains
Analysis of the HA1 gene of
the influenza A (H3N2)
hemagglutinin (HA) 5 amino
acid differences in all Texas
strains compared to the 200708 A/Wisconsin/67/2005
vaccine strain. All A/Texas
(H3N2) strains exhibited a
greater HA homology (99.099.7%) to the newly selected
2008-09 A/Brisbane/10/2007
strain.
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Vaccine Relatedness
Influenza B strains
All Texas strains were of the
B/Yamagata lineage and
genetically more homologous to
the 2008-09 B/Brisbane/5/2007
vaccine strain than the 2007/08
B/Malaysia/2506/2004 vaccine
strain.
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Antiviral Susceptibility
Adamantane. Matrix gene (MA) genetic analysis, specifically a serineto-asparagine substitution at position 31 (S31N), revealed adamantane
resistance in all influenza A (H3N2) strains but sensitivity in both
influenza A (H1N1) viruses.
Neuraminidase Inhibitors. All influenza A (H3N2) isolates were shown
to be sensitive to oseltamivir (Tamiflu) *. Genetic analysis of the
influenza B NA gene revealed that all Texas strains contained an
aspartic acid (D) residue at position 197 and are likely sensitive to
oseltamivir (Tamiflu).
*through genetic analysis of E119V, R292K, and N294S substitutions in the NA gene
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Family Transmissions
The inclusion criterion for household
transmission was defined as the onset
of influenza like illness ≥24 hours after
a confirmed index case.
HA1 hemagglutinin mutations
were observed within 3 of 7
families analyzed. This was
particularly noted within Family
12 (a-c), where 3 mutations
were observed in family
transmissions (Table 1).
Red Box denotes 2008/09 & 09/10 vaccine strain; Green asterisks (*) denote pediatric index cases.
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Family Transmissions
•3 of 7 families had at least 1 amino acid change
•Mutation at position 173 (within antibody binding site D) was
conserved within family transmissions, but highly variable
between families
HA1 Hemagglutinin Position in Family Transmissions
Strain
A/Texas/12a/2008
A/Texas/12b/2008
A/Texas/12c/2008
A/Texas/42d/2008
A/Texas/42a/2008
A/Texas/4a/2008
A/Texas/4a/2008
A/Brisbane/10/2007
3
L
F
L
F
F
L
L
L
83
K
N
K
N
N
K
K
K
123
E
E
E
E
G
E
E
E
173
E
E
E
N
N
Q
Q
K
208
R
R
R
R
R
R
G
R
300
I
V
I
I
I
I
I
I
Influenza Transmission Among Pediatric Patients and
Family Contacts
Results- Family Transmissions
173 Glu (E)
Polar, charged
173 Asn (N)
Polar, uncharged
173 Gln (Q)
Polar, uncharged
Three-dimensional depiction of the HA1 monomer Proteins
highlighting the variable mutation at position 173 observed
among families and the A/Brisbane Vaccine strain.
173 Lys (K)
Polar, charged
Influenza Transmission Among Pediatric Patients and
Family Contacts
Conclusions
PrimeMix™ real-time RT-PCR is more: 1) sensitive, 2) specific and 3)
timely compared to other detection methodologies including traditional
culture.
PrimeMix™ can detect influenza viruses at levels below the limits of
detection by culture.
Genetic analysis of the viruses from this cohort are a more optical match
to the current Brisbane vaccines strains.
The HA1 hemagglutinin protein is a highly evolving viral protein that can
mutate even among passage within a single human host.
Luke T. Daum, Ph.D.
QUESTIONS?
Longhorn Vaccines & Diagnostics
1747 Citadel Plaza, Suite 206
San Antonio, TX 78209
Phone: (210) 826-0910
Email: [email protected]

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