Eligibility requirements. Subjects were infants aged <12 months who were hospitalized for laboratory-confirmed influenza between October 2000 and April 2009 (prior to the arrival of the 2009 pandemic influenza in this region).
We excluded infants who were adopted at birth, infants whose mothers had a contraindication to inactivated influenza vaccine or were unable to consent to participate (eg, were deceased or had unknown whereabouts), infants who were hospitalized for reasons unrelated to respiratory infection (as determined by review of medical records), infants whose parents could not complete the interview in English or Spanish, and infants with influenza infection acquired when the patient was already in the hospital (ie, nosocomial). Infants who received influenza vaccine at least 2 weeks prior to admission to the hospital were excluded from the analyses, because it would be impossible to separate the effect of vaccination of the mother from that of vaccination of the infant.
Identification of potential cases.
Cases were infants hospitalized for influenza with documentation of either a nasal swab or aspirate sample that was positive for influenza by direct fluorescent antibody (DFA) test. Case subjects were identified from the list of all patients who had a nasal swab or aspirate sample submitted to the hospital's clinical virology laboratory for the DFA test (Light Diagnostics). This test has been shown to be 96.2% sensitive and 99.0% specific for influenza, compared with PCR, in our clinical virology laboratory [21
]. Samples deemed inadequate by the laboratory were not included. Data collection started in 2007. Subjects hospitalized between 2000 and early 2007 were identified historically from the clinical virology laboratory list of all tests for influenza and were enrolled by telephone. During the 2007–2008 and 2008–2009 influenza seasons, research staff identified cases prospectively by reviewing clinical virology laboratory lists of all tests for influenza and the daily list of new hospital admissions, to enroll patients in the hospital setting and to collect a nasal aspirate sample.
The nasal samples obtained from the case subjects identified prospectively (2007–2009) were all confirmed to be negative for 2009 pandemic influenza A H1N1. RNA was extracted from the clinical specimens using RNeasy Mini Kit (Qiagen) per the manufacturer's instructions. Reverse transcription and polymerase chain reaction were performed using primers and parameters described by the World Health Organization/CDC protocol [22
] and the AccessQuick RT-PCR System (Promega).
Selection of controls.
For each case, we enrolled 1 or 2 matched control subjects, who were hospitalized infants with DFA results negative for influenza. Control subjects from the list of all patients who had a DFA test for influenza were matched to cases by date of birth and date of hospitalization. Matching started with the subjects born within 2 weeks (before or after the case date of birth) and who were admitted within 2 weeks (before or after the case date of hospital admission) and then, if necessary, proceeded to those born within 4 weeks and admitted within 2 weeks from the case, then those born within 2 weeks and admitted within 4 weeks, and finally those born within 4 weeks and admitted within 4 weeks. We used lists of random numbers to determine the order in which to contact potential eligible subjects within each case-control group. We used risk-set sampling in our selection of cases and controls [23
Collection of data and ascertainment of vaccinations. We conducted interviews with the parents of all study subjects for information about demographic characteristics, possible confounders (such as breast-feeding or susceptible individuals in the household), and comorbidities and to identify all possible locations where vaccination was given. Interviews were conducted in person when a case subject or control subject was identified prior to discharge from the hospital, if possible, or otherwise by phone. All interviews were conducted in English or Spanish.
Information about vaccinations and comorbidities of the infants was obtained by reviewing records of all providers of medical care. We reviewed mothers' medical records from primary medical providers, obstetricians, pharmacies, and anywhere the mother stated that she had received influenza vaccine. We used this information to ascertain whether a woman had received influenza vaccine during pregnancy, whether she had received the vaccine at any time prior to that pregnancy, and whether she had received the vaccine during the same influenza season as the infant's hospital admission. A woman was considered vaccinated if there was written documentation of receipt of influenza vaccine during her pregnancy, excluding vaccinations received within 14 days of delivery.
We also collected clinical data from the hospital medical record, including any abnormal vital signs (highest temperature and respiratory rates and lowest oxygen saturation levels), clinical signs of increased work of breathing, results of chest radiographs, and the need for intubation and/or admission to the intensive care unit (ICU). We classified the severity of each case subject's symptoms on a scale of 0–16 points, based on our modification of a validated scale of severity of respiratory symptoms in infants () [24
Clinical Severity Scale Used to Assess Severity of Influenza
Statistical analysis. We calculated a matched odds ratio for vaccination of mothers of case subjects, compared with mothers of matched control subjects. The vaccine's effectiveness was calculated as 1 minus the matched odds ratio, multiplied by 100. Conditional logistic regression was used to adjust for potential confounders, including race, ethnicity, sex, age, day care attendance, prematurity, vaccination of household contacts, breast-feeding, and relevant chronic illness (asthma/reactive airways disease, chronic lung disease, conditions requiring medical equipment to facilitate breathing, heart defects, blood disorders, seizures, metabolic or endocrine disorders, severe gastrointestinal disease, kidney disease, or spinal cord injury).
A stratified analysis was also conducted to assess for effect modification by age of the subject (
6 months vs <6 months) on the basis of the CDC recommendation to begin influenza vaccination at age 6 months. Whether the subject was identified at the time of hospitalization or historically via billing data was also evaluated as a possible confounder or effect modifier.
We also assessed the significance of the clinical severity of influenza of the cases using Student's t test or the Wilcoxon rank-sum test, as appropriate. Analyses were conducted using SAS, version 9.1.3 for Microsoft Windows (SAS Institute).