2a. Study Design
To compare adult hospitalization rates for laboratory-confirmed influenza in two geographically distinct Tennessee counties, we enrolled adults ≥50 years hospitalized with respiratory symptoms or non-localizing fever at two hospitals in each county during the influenza season. Recruitment occurred from November 2006 through April 2007 beginning two days per week. Once two cases of influenza were identified for two consecutive weeks in the hospital laboratories in each county, surveillance increased to 4-7 days per week in the county. IRB approval was obtained from all participating hospitals.
2b. Pediatric School-Based Program
In the fall of 2006, Knox County Department of Health implemented a school-based immunization program in which live intranasal attenuated influenza vaccine was administered (LAIV, MedImmune, Gaithersburg, MD), similar to the previously reported 2005-2006 campaign.[7
] Between September and December 2006, 47% of 54,786 public school students and 61% of 5,998 private school children in Knox County were vaccinated. This represented 61% of elementary students, 45% of middle school students and 26% of high school students (John Lott, personal communication). In children aged <9 years, 53% received the recommended second dose of LAIV. Davidson County did not have such a school-based immunization program.
2c. Study Population
All adults ≥50 years with acute respiratory symptoms or non-localizing fever admitted to one of four hospitals on surveillance days were eligible for enrollment. During that period Davidson County had a population of 575,261 individuals with 51.3% women, 67.8% white, 27.5% black, while Knox County (180 miles east of Davidson County) had a population of 404,972 individuals with 51.4% women, 88.2% white, and 8.7% black. Both counties are urban with surrounding suburban and rural counties. Davidson County surveillance hospitals included one academic medical center, and one community hospital; Knox County hospitals included two community hospitals. Eligible adults, residing in the surveillance county, were those admitted during specific 24-hour surveillance periods for each enrollment day (4 to 7 days per week during influenza season) and had one or more of the following admitting symptoms: cough, fever, nasal congestion/coryza, dyspnea (including cardiac causes), or wheezing. All eligible patients were entered into a screening log that included age, sex, race, insurance status, admission symptoms, and provision of informed consent.
2d. Demographic and Clinical Information
Patient questionnaires and chart review data collection instruments captured Centers for Disease Control and Prevention (CDC)-defined high risk conditions,[8
] living conditions, clinical symptoms, past medical history, smoking history, use of specific medications (steroids, immunosuppressants, and home oxygen), and influenza vaccination status for the 2006-2007 season. Results of provider-ordered microbiologic and radiographic tests, hospital course (ICU stay, length of hospitalization, intubation, and oxygen use), disposition at discharge, and discharge diagnoses were captured through chart review.
2e. Verification of Influenza Vaccination of Adults
Enrolled adults were asked if they received an influenza vaccine for the current influenza season. Verification of vaccination status in all adults was attempted by contacting the provider; vaccination records were considered the gold standard. However, many adults received their influenza vaccinations from non-traditional providers such as grocery chains and pharmacies, thus we were unable to verify vaccination from these sites. Three vaccination categories were defined: not vaccinated (included both verified and unverified patient-reported non-vaccination as well as those vaccinated within 14 days of hospitalization); verified, vaccinated and not verified, vaccinated. Influenza negative patients were used to estimate the rate of influenza vaccine uptake among county adults.
2f. Laboratory Methods
After informed consent, nasal and throat swabs were obtained. One swab sampled both nostrils, a second swab sampled the posterior pharynx, and both swabs were placed into Hank’s transport media. Specimens were held at 4°C, divided into multiple aliquots within 24 hours, and stored at -80°C. Aliquots stored in lysis buffer (Roche Applied Science, Indianapolis, IN) were used for influenza A and B reverse transcriptase-polymerase chain reaction (RT-PCR). Specimens that were influenza positive by RT-PCR were cultured for influenza typing.
Using standard molecular biologic protocols (Roche Applied Science), RNA was extracted from the frozen aliquots and real-time RT-PCR assays were performed using primers and probes developed and provided by Steve Lindstrom, Influenza Branch, CDC, Atlanta, GA.[9
] All samples were tested for β-actin (Applied Biosystems) to insure specimen quality. If β-actin was absent in three consecutive tests on a sample, the RT-PCR results were categorized as indeterminate. Influenza was confirmed by positive RT-PCR on two separate tests.
Viral cultures were performed on influenza RT-PCR positive specimens using Rhesus monkey kidney cell culture tubes (Diagnostic Hybrids). Hemadsorption with guinea pig red blood cells was used to screen isolates and influenza A or B was confirmed using specific monoclonal antibodies (Viromed). Influenza isolates from study subjects were then sent to the CDC World Health Organization Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, GA for typing.
2g. Statistical Analysis
The primary study objective was to compare population-based rates of influenza-associated hospitalizations in adults aged ≥50 years living in two Tennessee counties, one with a school-based immunization program (Knox) and the other without such a program (Davidson). Rates of influenza-associated hospitalizations were defined as the seasonal weighted number of hospitalizations attributable to influenza-related acute respiratory tract infections or non-localizing fever divided by the county population according to 2006 U.S. census estimates, multiplied by 1000. The observed number of enrolled hospitalizations was weighed to account for sampling days, number of eligible patients, and hospital market share (defined below). Adults with indeterminate PCR results were considered eligible but non-enrolled. Rates were calculated for adults 50-64 years and ≥65 years of age for each county and 95% confidence intervals and rate differences were determined using 1000 bootstrap samples. P values of rate comparisons were determined by the widest confidence interval excluding zero using the 1000 bootstrap samples.
Since we assumed that school-based immunization would have a major effect on county disease rates, this study was powered to detect a 50% lower rate in the intervention than the control county. In low and moderate influenza seasons with projected influenza hospitalization rates of 1.29 and 2.57 per 1000 population, respectively, we projected >80% power to detect relative rates of 0.39 and 0.55 in the intervention county.
2h. Market Share
The Tennessee Hospital Discharge Database, an electronic database that reports all hospitalizations and emergency department discharges in the state, was used to establish where Davidson and Knox County residents with ARI or non-localizing fever were admitted, and what proportion of those admissions occurred in the surveillance hospitals in each county. Specific International Classification of Diseases version 9 (ICD-9) codes used to define ARI or non-localizing fever included: 036.2, 038, 079.9, 320, 321, 323, 381-383, 410, 411, 413, 428, 460-466, 480-487, 490-494, 496, 507, 510-514, 518.81, 780.6, 771.8, 786, 790.7, 799.02,995.91 and 995.92.