1. Influenza Transmission Among Pediatric Patients and Family Contacts 1747 Citadel Plaza Suite 206 San Antonio, TX 78209 (210) 826-0910 Longhorn-VandD@sbcglobal.net

2. Background Influenza virus: a highly evolving pathogen- In the US: 200K hospitalizations, 35,000 deaths per yr.200K hospitalizations, 35,000 deaths per yr. 72 pediatric deaths in 2007/0872 pediatric deaths in 2007/08 Increased drug resistance (Tamiflu and adamanatane)Increased drug resistance (Tamiflu and adamanatane) Worldwide (as of July 6 th ):Worldwide (as of July 6 th ): 436 H5N1 bird flu cases, 262 deaths (61% fatality)436 H5N1 bird flu cases, 262 deaths (61% fatality) 94K H1 swine cases, 429 deaths (0.45% fatality)94K H1 swine cases, 429 deaths (0.45% fatality) Influenza Transmission Among Pediatric Patients and Family Contacts

3. 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 Influenza Transmission Among Pediatric Patients and Family Contacts COLLECTKILLSTABILIZEPRESERVE DISPENSEADD NUCLEICSANALYZE

4. Specific Aims Influenza Transmission Among Pediatric Patients and Family Contacts 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.

5. Experimental Design Influenza Transmission Among Pediatric Patients and Family Contacts 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 PrimeStore TM Solution and extracted using the Ambion RNAqueous Micro Kit. Real-time RT-PCR was performed using PrimeMix TM Solution. Genetic characterization of selected clinical samples preserved in PrimeStore™ was performed using standard RT-PCR and nucleotide sequencing.

6. Experimental Design Influenza Transmission Among Pediatric Patients and Family Contacts Criteria for Patient Enrollment 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

7. Results Influenza Transmission Among Pediatric Patients and Family Contacts 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

8. Results- Sensitivity and Specificity Influenza Transmission Among Pediatric Patients and Family Contacts Detection Summary by Method (N=222) TypeSubtype FluAFluBH3H1 (human) Rapid Antigen3312NA Culture39*17NA PrimeMix RT-PCR4719442 *One reported culture-positive was not detected by PrimeMix

9. Results- Sensitivity and Specificity Influenza Transmission Among Pediatric Patients and Family Contacts

10. 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- Sensitivity and Specificity

11. Results- Vaccine Relatedness Influenza Transmission Among Pediatric Patients and Family Contacts 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 2007- 08 A/Wisconsin/67/2005 vaccine strain. All A/Texas (H3N2) strains exhibited a greater HA homology (99.0- 99.7%) to the newly selected 2008-09 A/Brisbane/10/2007 strain.

12. Results- Vaccine Relatedness Influenza Transmission Among Pediatric Patients and Family Contacts 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 B strains

13. Results- Antiviral Susceptibility Influenza Transmission Among Pediatric Patients and Family Contacts Adamantane. Matrix gene (MA) genetic analysis, specifically a serine- to-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

14. Results- Family Transmissions Influenza Transmission Among Pediatric Patients and Family Contacts 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). The inclusion criterion for household transmission was defined as the onset of influenza like illness ≥24 hours after a confirmed index case. Red Box denotes 2008/09 & 09/10 vaccine strain; Green asterisks (*) denote pediatric index cases.

15. Results- Family Transmissions Influenza Transmission Among Pediatric Patients and Family Contacts 3 of 7 families had at least 1 amino acid change 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 Mutation at position 173 (within antibody binding site D) was conserved within family transmissions, but highly variable between families Strain HA1 Hemagglutinin Position in Family Transmissions 383123173208300 A/Texas/12a/2008LKEERI A/Texas/12b/2008FNEERV A/Texas/12c/2008LKEERI A/Texas/42d/2008FNENRI A/Texas/42a/2008FNGNRI A/Texas/4a/2008LKEQRI LKEQGI A/Brisbane/10/2007LKEKRI

16. Results- Family Transmissions Influenza Transmission Among Pediatric Patients and Family Contacts 173 Glu (E) Polar, charged 173 Asn (N) Polar, uncharged 173 Gln (Q) Polar, uncharged 173 Lys (K) Polar, charged Three-dimensional depiction of the HA1 monomer Proteins highlighting the variable mutation at position 173 observed among families and the A/Brisbane Vaccine strain.

17. Conclusions Influenza Transmission Among Pediatric Patients and Family Contacts  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.

18. Longhorn Vaccines & Diagnostics 1747 Citadel Plaza, Suite 206 San Antonio, TX 78209 Phone: (210) 826-0910 Email: Longhorn-VandD@sbcglobal.net Longhorn-VandD@sbcglobal.net Luke T. Daum, Ph.D. QUESTIONS?