Post-hoc, secondary analyses were performed on data from healthy volunteers enrolled in an influenza challenge study conducted at the National Institutes of Health (NIH) Clinical Center. Study design and primary results were reported previously and adheres to CONSORT guidelines for reporting clinical trials as applicable . Briefly, the study investigated whether participants with a high pre-challenge serum HAI titer were less likely to develop mild to moderate influenza disease after intranasal inoculation with a wild-type influenza A/H1N1pdm virus compared to participants with a low pre-challenge HAI titer.
After signing written informed consent, 65 healthy men and women between 18 to 40 years of age underwent a 9-day inpatient quarantine and intranasal challenge via a nasal atomizer with an infective dose of H1N1pdm and were then monitored in an outpatient clinic periodically over 2 months. Participants were blinded to viral shedding and antibody titer results. Participants received no antiviral medications during the first 7 days post-inoculation and a small number were given other medications to control symptoms if necessary. Participants were tested at baseline to rule out the presence of other viral pathogens. Participants completed the FLU-PRO questionnaire on paper the evening of Day − 1 and morning of Day 0 (pre-inoculation), evening of Day 0 post-inoculation, and twice daily thereafter to Day 14 post-inoculation and then once more on Day 28. The FLU-PRO is a 32-item daily diary assessing influenza signs and symptoms across 6 body systems: Nose (4 items), Throat (3 items), Eyes (3 items), Chest/Respiratory (7 items), Gastrointestinal (4 items), and Body/Systemic (11 items). Respondents are asked to rate each sign or symptom on a 5-point ordinal scale, with higher scores indicating a more frequent sign or symptom. For 27 of the items, the scale is as follows: 0 (“Not at all”), 1 (“A little bit”), 2 (“Somewhat”), 3 (“Quite a bit”), and 4 (“Very much”). For 5 items, severity is assessed in terms of numerical frequency, i.e., vomiting or diarrhea (0 times, 1 time, 2 times, 3 times, or 4 or more times); with frequency of sneezing, coughing, and coughed up mucus or phlegm evaluated on a scale from 0 (“Never”) to 4 (“Always”).
The FLU-PRO total score is computed as a mean score across all 32 items comprising the instrument. Total scores can range from 0 (symptom free) to 4 (very severe symptoms). Six domain scores are also computed, representing symptom severity in each of the assessed body areas. Each domain score is calculated as the mean of all items comprising the domain, with scores ranging from 0 to 4.
The FLU-PRO has been tested as a once-daily measure . In this study, participants completed the diary twice a day. Using paired t-tests, no significant domain or total score differences were observed between the two pre-inoculation administrations (evening Day − 1 and morning Day 0) or the mornings and evenings of Days 1 through 14 post-inoculation (data not shown). Given no added discrimination of a twice daily administration and to be consistent with current use, a daily mean FLU-PRO score was calculated for each participant, taking the average of scores from the two pre-inoculation administrations, and the morning and evening administration of each day post-inoculation. All subsequent analyses used mean daily scores.
Analyses were performed on symptomatic participants only, with asymptomatic participants defined by a FLU-PRO daily total score of zero at every study assessment (Day − 1 to Day 14, and Day 28). Descriptive statistics were used to characterize demographics and clinical characteristics of the study population.
The distributional characteristics of FLU-PRO domain and total scores, including frequency of symptom occurrence within the sample (n, %) and severity of symptoms (score mean (standard deviation, SD), range, floor, ceiling) were examined pre-inoculation, on Day 3 (peak viral shedding and peak number of symptoms), and on Day 10 post-inoculation (by which time most participants were discharged). These days were selected based on viral shedding and number of clinician-assessed symptoms as reported by Memoli et al. . The trajectory of FLU-PRO domain and total scores from Days − 1 to 10 were also plotted and examined.
Analysis of variance (ANOVA) was used to test the ability of FLU-PRO severity scores to differentiate two or more clinical groups. Specifically, domain and total scores on Day 3 were compared by viral shedding status (shedding vs. no shedding), baseline HAI (high vs. low titers), baseline NAI (high vs. low titers), and the combination of baseline HAI-NAI titers (high HAI-high NAI vs. low HAI-high NAI vs. low HAI-low NAI). Day 3 was selected as it was the day of peak viral shedding and peak number of symptoms.
For this set of analyses, assuming the differences between these known-groups would be similar to the difference between influenza positive patients with no/mild symptoms [Mean = 0.98 (SD 0.47)] and moderate symptoms [1.38 (SD 0.57)] , the power for detecting significant differences between these known-groups was 0.97.