In November and December 2009, after pH1N1 virus activity in Tampa Bay had peaked (), we collected a convenience sample of de-identified, leftover serum specimens (initially drawn for other laboratory testing) from residents of Pasco, Hillsborough, Manatee and Pinellas counties in Tampa Bay, Florida.
We sought to collect 160 specimens from each of six age groups: <5 years, 5–17 years, 18–24 years, 25–49 years, 50–64 years and ≥65 years. Infants less than 6 months were excluded due to the potential for maternal antibody transmission. The required sample size was calculated using relative standard error measurements. We estimated that the lowest seroprevalence for all groups would be among adults aged ≥65 years. At the time of the serosurvey, we estimated that 15% of this age group would be seropositive, requiring a sample size of approximately 160 to maintain a relative standard error less than 20%. We were able to collect at least 160 samples from all ages groups except for children aged <5 years for which we were only able to collect 60 samples. We estimated that the seroprevalence among this age group would be high (30%), and therefore despite the small sample size, would meet the relative standard error criteria of less than 20%.
This study was proposed to the Florida Department of Health Institutional Review Board (IRB) and Centers for Disease Control and Prevention (CDC) IRB who considered the investigation as public health response, and therefore not subject to IRB review and approval.
Leftover serum specimens for Tampa Bay residents aged ≥16 years were collected from a large blood bank testing facility during a 4-day period from November 30 -December 3, 2009 (). For residents aged <16 years, leftover specimens were collected from a children's medical center and an outpatient pediatric clinic from November 14 to December 31, 2009. The majority of specimens collected from the children's medical center had been collected for allergy and immunology testing. Leftover specimens from the pediatric clinic had been originally collected for routine outpatient testing.
Antibodies against pH1N1 virus were detected by the hemagglutination-inhibition (HI) assay as previously described using A/California/07/2009 virus 
. All specimens were tested in triplicate. Specimens with a geometric mean HI titer ≥40 were considered to be seropositive. Total seroprevalence results were age-standardized based on Tampa Bay population estimates from the American Community Survey. Previous studies have shown that an HI titer ≥40 is associated with a ≥ 50% reduced risk of contracting seasonal influenza virus infection among susceptible persons 
Using sera from patients with pH1N1 laboratory-confirmed infections and non-exposed United States residents, a previous study determined that a threshold HI titer ≥40 yielded a sensitivity of 75% and specificity of 97% in determining previous infection with the pH1N1 virus among persons <60 years of age 
; specificity was shown to decrease to 94% among persons >60 years of age. We adjusted the overall seroprevalence results to account for both the sensitivity and specificity of the HI assay, terming the resultant estimate the assay-adjusted seroprevalence (Appendix S1
Because serology cannot differentiate between antibodies produced by virus infection and response to vaccination, we developed a simple statistical model to estimate the proportion of seropositive results due to pH1N1 vaccination coverage (i.e., vaccine-induced seropositivity ) (Appendix S2
). Monthly vaccination coverage estimates for the state of Florida during November and December 2009 were calculated based on combined Behavioral Risk Factors Surveillance System (BRFSS) and National 2009 H1N1 Flu Survey (NHFS) data 
. In the model we used vaccination coverage estimates, vaccine immunogenicity estimates from the literature 
, and an estimate of the proportion infected prior to vaccination to estimate the proportion with vaccine-induced seropositivity not infected prior to vaccination. To estimate the proportion of the population infected with the pH1N1 virus prior to the serosurvey, we subtracted this proportion from the seroprevalence estimate.