Discussion
We compared aerosol RNA shedding in influenza A cases infected naturally and by nasal instillation under experimental conditions. Previously, we cultured influenza virus from exhaled breath showing that quantitative culture correlates with RNA copy detection (r = 0.34, p<0.0001).15 A minority of experimental cases shed virus into aerosols (28%). A far greater proportion of the naturally infected study population shed into aerosols (86%). Among the experimentally infected with detectable viral aerosols, the fine RNA copy GM was within log10 that for naturally infected cases. A more substantial difference in fine aerosol shedding rate was observed at the level of the overall distribution, with increases in median and upper percentiles for naturally infected cases (Figure S3). Compared with naturally infected cases at the 95thpercentile of fine aerosol shedding, experimentally infected cases shed nearly 2.5 log10 fewer RNA copies. Given the selection of naturally infected cases on ILI symptoms and/or a positive rapid antigen test, and an observed relationship between symptoms and shedding (Table 3), it is possible that aerosol shedding observed for the naturally infected study populations overestimates what might be expected for a sample representative of all naturally-acquired infections.
Peak RNA copies shed into aerosols on day 3 post nasal inoculation was one day later than that observed in upper respiratory mucosa in previous challenge studies,7 yet consistent with the 1-2 day post symptom onset nose and throat swab viral load peak from household contact surveillance studies.8 There are no available data with which to compare the current study’s aerosol shedding. If we take previous estimates of ~1-2 days as the influenza A incubation period, plus about 1 day post symptom onset to reach peak aerosol shedding in the naturally infected population, then we would expect peak aerosol shedding on ~2-3 days following exposure to virus. This is consistent with the peak in aerosol shedding in the experimentally infected cases at 3 days post inoculation, suggesting that the progression of infection from exposure to replication is consistent with the natural infection group and with other studies. The significant decrease in aerosol shedding by day post onset (Table 3) among the naturally infected, but not the experimental cases, could be related to failure to detect a clear trend against a background of very much lower overall aerosol shedding for the experimental group. The temporal decline observed for naturally infected cases along with previous reports of less temporal decline in viral load from nasopharyngeal swabs compared with fine aerosols following day 1 post symptom onset,15 and a tendency for nasal viral load to overestimate transmission risk after day 3 post symptom onset,12 suggests aerosol shedding may better fit epidemiologically observed transmission dynamics over time in the household setting.
Lower respiratory and cough symptom scores were associated with viral aerosol detection in both groups with a clear dose response relationship in cough score for the naturally infected cases, supporting the notion of a symptomatology-shedding relationship for aerosols. Peak symptom scores and aerosol shedding coincided, consistent with the temporal dynamics of other studies.7–9 However, regression analyses that restricted observations to maximum fine aerosol samples found mostly weak and unstable effects of symptoms on shedding rate. Only body temperature in the combined population analysis was significantly associated with shedding strength. These data should be interpreted with caution given limited heterogeneity in symptom severity, with milder illness characteristic of the experimental cases and more moderate to severe illness characteristic of the naturally infected cases. It may be for this reason that other studies have reported mixed results with respect to associations between symptomatology and nose/throat viral load.9,10 The current data may nevertheless indicate that febrile periods of illness are associated with increased infectivity risk. Studying cases with a broader range of symptom severity could provide additional insight into symptom-shedding relationships with useful implications for identifying contagious symptom profiles.
There is growing evidence that airborne transmission plays an important role in the spread of influenza.5,14,20,21 Humans experimentally challenged to influenza virus by airborne particles had a 50% risk of infection to a 0.6-3.5 TCID50 dose and exhibited increased propensity for moderate to severe illness with fever and cough compared with others experimentally challenged by nasal droplets.1,2 The term anisotropic has been used to describe such infections where inoculation mode determines illness presentation.4,5 A population of cases with naturally-acquired infections would be expected to demonstrate a higher proportion of moderate-severe influenza-like illness compared with a population of cases exclusively infected by exposure to the nasal mucosa. Compared with other symptoms, systemic scores declined more rapidly following day 1 post symptom onset for natural cases, consistent with other findings8 and suggestive of the immune system clearing systemic infection. These findings may hint that natural cases may be more likely to result in lung and systemic infection initiated by an airborne dose, whereas experimentally infected cases with only nasal mucosal exposure were more likely to have few if any systemic symptoms and illness more localized to the upper respiratory tract.
Given the selection bias and the contrasted symptom profiles in two populations and observed associations between symptoms and shedding, we attempted to adjust for the effect of symptoms to understand the direct effect of experimental versus natural infection on the viral load in fine particle aerosols (Figure S1). Ultimately, propensity score modelling failed to balance the distribution of covariates between groups and we concluded that the groups were simply too different to achieve an unbiased estimate of the main effect of group membership (experimental versus natural) on shedding strength. Assuming minimal contribution of potential confounders on the pathway between mode of inoculation and study population membership (i.e., age, sex, host immunity, virus pathogenicity, dose) we cannot conclude that the unadjusted differences in symptomatology and shedding are a result of mode of inoculation, or simply the result of the differences inherent in recruitment and enrollment procedures and other potentially unobserved confounders in the absence of a randomized controlled design.
Identifying naturally infected reference groups that represent the true distribution of symptom severity presents a challenge. Although a substantial proportion of cases are asymptomatic, symptomatic community cases are more prone for inclusion in epidemiologic studies upon seeking medical attention.22 Multi-year sero-surveillance of large cohorts in the UK shows influenza infections presented asymptomatically at a rate of 77 per 100 person-seasons.23 A meta-analysis of longitudinal studies using serological evidence of infection and controlling for background illness reported a 65%-85% asymptomatic fraction.24 It is possible that the experimentally infected cases are not substantially different in symptomatology from a representative sample of all influenza infections. Accessing a representative sample might be achieved through intensive household or dormitory surveillance of contacts of known cases.
The infectious dose for airborne influenza, and the infectious potential of cases infected by various modes is largely unknown. If the typical fine aerosol shedding rate from influenza cases is important for driving airborne transmission, then our findings would indicate that nasal mucosal exposure in the experimental challenge model produces cases with airborne infectious potential similar to symptomatic cases infected naturally by contact, large droplets, or fine aerosols. If above average shedders are important for driving airborne transmission (i.e., superspreader hypothesis), then infections acquired through nasal mucosa may not pose as much airborne infectious potential. If we assume hypothetically that the symptomatic naturally infected cases drawn from the University of Maryland campus community represent the upper 1% of symptom severity and shedding strength in a broader population, and if we also assume that the experimental cases are representative of total community infections, the chances of an experimental case reaching the level of fine aerosol shedding observed in the naturally infected group would be 0.39% (1% of 39 experimental cases). If shedders in the upper percentiles of shedding rate are responsible for driving transmission, then it would take many more experimental cases to adequately simulate transmission events in a human transmission challenge trial model. This introduces logistical challenges and motivates work to identify, among naturally infected shedders, characteristics predictive of aerosols (and mucosal) shedding. In particular, response to infection by different modes may vary between children and adults, with implications for subsequent infectivity and population epidemiology.10,13,25,26 Clinical detection of infections that may be associated with disease severity and potential for self-isolation or other behaviors that could modify contagiousness should be considered in population-level transmission risk assessment.
Given bias introduced by selection of natural infections on symptoms plus fever or rapid antigen test, the observed correlations of symptoms and fever with viral shedding into aerosols, and a small N with minimal covariable distributional overlap precluding appropriate adjustment, we conclude that the naturally infected population is too different from the experimentally infected cases to make valid comparisons. Our observations show that the 52 nasally inoculated experimental cases produced viral aerosol shedders less frequently than the 83 symptomatic naturally infected population. When they did shed detectable virus into aerosols, the experimental cases did so at substantially lower quantities than the symptomatic naturally infected group. This difference in aerosol shedding was most pronounced when comparing the highest percentiles of aerosol shedding for each group. The probability and quantity of aerosol shedding in unselected natural infection is unknown. Therefore, these findings encourage efforts to evaluate shedding from infections observed during contact surveillance without selection based on symptoms or fever.