Results
For 39 experimental and 83 naturally infected influenza A H3 cases, there were 84 and 146 exhaled breath collection instances respectively. Of the 39 confirmed experimental cases, 36 were qRT-PCR positive two or more days, 31 of whom also had serological evidence of infection; three were qRT-PCR positive on one day only and had serological evidence of infection. A total of 52 challenge study volunteers were inoculated, giving an infection rate of 75%, based on the current case infection definition.
Both study populations of young adults were generally healthy. The experimental group was on average 10 years older than the naturally infected. Experimental cases were more likely to be male while naturally infected cases were balanced by sex (Table 1). Experimental cases had illness mostly characterized by upper respiratory symptoms, or were asymptomatic (12.8%) (Table 1). There were small peaks in upper respiratory, lower respiratory, systemic, and cough scores, and cough counts on day 3 post inoculation. Naturally infected cases had more severe symptoms scores and greater cough counts, with symptom scores peaking on day 1 post symptom onset and aligning with day 3 post inoculation (Figure 1).
The risk of shedding virus into coarse and fine aerosols for experimentally infected was 6/39 (15%) and 11/39 (28%), and for naturally infected 45/83 (54%) and 71/83 (86%) (Table 2, Figure S4a). Median coarse and fine aerosol shedding quantity between the groups was significantly higher for natural cases in both fine (p<0.001). and coarse (p<0.001) aerosol fractions. Peak aerosol shedding was observed on day 3 post inoculation and day 1 post symptom onset for experimental and natural cases, respectively, matching peak symptom score day alignment (Figure 2). No virus was detected in fine aerosols on day 1 post inoculation in experimental cases. On the day of peak aerosol shedding, median symptom scores for experimental infection were upper respiratory 4 (IQR 2, 5), lower respiratory 0 (0, 1), systemic 1 (0, 2), cough 0 (0, 1), and cough count 0 (0, 6) and for natural infections 7 (5, 9), 3 (2, 4), 6 (4, 8), 2 (2, 3), and 22 (8, 40), respectively.
When using all the detectable qRT-PCR nasopharyngeal swab samples from days 1-6 post inoculation (N=179) and 1-3 post symptom onset (N=143), Ct values were notably lower for natural compared with experimental cases (Figure S2). The lowest Ct values were seen on day-1 post symptom onset in naturally infected cases and day-3 post inoculation; subsequent days showed a faster rise in Ct for experimental compared with natural cases.
Restricted to maximum shedding observations by aerosol fraction, the GM (GSD) for coarse and fine aerosols was 2.7E+3 (3.3) and 5.1E+3 (4.7) for experimentally, and 2.1E+4 (16.5) and 5.1E+4 (17.0) for naturally infected cases (Table 2, Figure S3). Descriptive statistics for covariates during maximum fine aerosol shedding observation days (Table S3) are similar to those derived from all observation days. Upper respiratory symptoms score distributions overlapped the most between groups, while differences in the distributions of lower respiratory, systemic, and cough symptoms scores, and cough count were more pronounced (Figure 3).
For the experimental cases, unadjusted upper and lower respiratory scores, cough symptoms, and cough count were positively associated with fine aerosol shedding detection (Table 3). For naturally infected cases, unadjusted lower respiratory symptom scores, and cough symptoms were positively associated with fine aerosol shedding risk. All symptom related covariates were associated with aerosol shedding risk after combining both populations. Study day was negatively associated with aerosol shedding in naturally infected cases. Nasopharyngeal swab qRT-PCR cycle threshold (Ct) value had a negative association with aerosol shedding risk for both groups, separately and combined. There were no significant predictors of aerosol shedding rate among experimental or natural cases, and only after combing groups did body temperature reach a significant association. Of the experimentally infected, only males shed detectable virus into aerosols.
After extensive testing, the best propensity score model (covariates: fever >37.8oC, body temperature, and upper respiratory symptom score) and adjustment by inverse probability weighting for average treatment effect (ATE) minimized the standardized differences between the groups with mean absolute value standardized difference of 91 (Table S5). After ATE adjustment, balance improved for some covariates, however not to the point where they could be considered similar (Figure 4, Table S5). Although it is advisable that absolute standardized differences for covariates not exceed 10% between comparison groups, and variance ratios approach one (range 0.5-2),18 the best model with weighted adjustment had absolute standardized differences ranging 7.3% to 169.1% and variance ratios up to 48.5. The substantial differences between covariate distributions did not support the use of propensity score adjusted approaches for making further comparisons between these populations.