Humidity as a non-pharmaceutical intervention for influenza A

Influenza is a global problem infecting 5–10% of adults and 20–30% of children annually. Non-pharmaceutical interventions (NPIs) are attractive approaches to complement vaccination in the prevention and reduction of influenza. Strong cyclical reduction of absolute humidity has been associated with influenza outbreaks in temperate climates. This study tested the hypothesis that raising absolute humidity above seasonal lows would impact influenza virus survival and transmission in a key source of influenza virus distribution, a community school. Air samples and objects handled by students (e.g. blocks and markers) were collected from preschool classrooms. All samples were processed and PCR used to determine the presence of influenza virus and its amount. Additionally samples were tested for their ability to infect cells in cultures. We observed a significant reduction (p < 0.05) in the total number of influenza A virus positive samples (air and fomite) and viral genome copies upon humidification as compared to control rooms. This suggests the future potential of artificial humidification as a possible strategy to control influenza outbreaks in temperate climates. There were 2.3 times as many ILI cases in the control rooms compared to the humidified rooms, and whether there is a causal relationship, and its direction between the number of cases and levels of influenza virus in the rooms is not known. Additional research is required, but this is the first prospective study suggesting that exogenous humidification could serve as a scalable NPI for influenza or other viral outbreaks.

* Corresponding author, e m a i l : Pierret.Christopher@mayo.edu ( C P ) 2 9 3 0 ¶ These authors contributed equally to this work. The number of positive samples in each sample type is indicated, the total number of samples  (NLDAS) project [20]. Absolute humidity was calculated using Excel software using formulas as 2 5 0 previously described [16]. NIOSH two-stage bioaerosol cyclone samplers [28,29] collected air samples and separated them  After class dismissal, particle counts were measured in the center of each classroom at a height Lake Forest, IL). Sizes of particles measured were 0.3, 0.5, 1, 2.5, 5, and 10 μ m.

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Particle size data was binned into sizes to match those collected by the NIOSH samplers such and 10 μ m. Particle counts were also measured before and after humidifier turned on in a pilot classroom were also wrapped including rolling pins, hard rubber brayers, and plastic pizza 2 8 7 cutters. After play, paper was transported back to BSL2 laboratory. Processing of fomites and air samples 2 9 0 Papers from classroom objects were lightly dusted with fingerprinting powder (Hi-Fi Volcano Latent Print Powder, Sirchie Youngsville, NC). Once fingerprint was identified, a portion of 2 9 2 paper (~3.5-4 cm 2) was removed and placed into a 15 mL conical tube containing 1 mL of 2 9 3 infection media. Samples were placed on ice until ready for further processing. Both fomites (paper) and air samples (in media) were vortexed briefly and incubated on ice for 2 9 6 15 minutes. Samples were vortexed again prior to centrifugation for 20 minutes at 4 °C at 3313g 2 9 7 (15 mL tubes) and centrifuged at room temperature at 1520g in a microcentrifuge (1.5 mL 2 9 8 tubes). Liquid was removed from 15 mL conical tubes and transferred to 1.5 mL conical tubes. The supernatant from these samples was frozen at -80 °C and used for subsequent RNA isolation  Clinic, Rochester, MN. Samples were thawed and used to infect bulk cultures of MDCK. run on an xCELLigence RTCA MP instrument (ACEA Biosciences, Inc., Sand Diego, CA).

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After calibration with media, wells were seeded with MDCK cells. Twenty-four hours later,  Readings were taken every 15 min for 7 days post inoculation. Using influenza A virus stock,  isolation with the Viral RNA isolation kit (Qiagen) as per the manufacturer's instructions, Quantitect kit). The PCR thermal profile consisted of an initial cDNA step of 30 minutes at 50 and 30 seconds at 72 °C. Detection, quantification and data analysis were performed in the CFX 3 2 9 manager real-time detection system (Bio-Rad).

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Though Influenza A and B and RSV were included in this analysis, low numbers of positive primers containing T7 promoter sequence in the forward sites: Inf AF: 5′- converted to molecular copies according to the formula: The detection limit of the SYBR green real time RT-PCR assay was determined by testing serial 3 4 9 ten-fold dilutions of the in vitro transcribed influenza A viral RNA ranging from 10 1 to 3 5 0 10 7 copies/µL. Cycle-threshold (Ct) values were plotted against the RNA copy number to Using serially diluted in vitro transcribed influenza A RNA, viral particles were detectable 3 5 7 ranging from 10 1 -10 7 RNA copies using the described assay. qRT-PCR sampling was 3 5 8 sufficiently sensitive to detect as few as 7 RNA copies. Percentage of samples positive for influenza A ( control rooms, with a 95% confidence interval and p-value. Odds ratios below one (with 3 6 5 statistical significance) demonstrate protective effects of humidification in that positive influenza was less likely to be obtained by the relevant capture system. Capture systems for influenza the paper and air capture systems. Mean copy number of influenza A positive samples (Table S1): To account for within-room humidified rooms as compared to control rooms, with a 95% confidence interval and p-value.  The data that support the findings of this study are available from the corresponding author upon 3 7 9 request.    MEMH directed UMR undergraduate work-study students in processing of paper wrapped 4 1 2 objects. HBL assisted with air particle analyses. JLG analyzed student absence/attendance data. manuscript. WCH provided wording for collecting ILI school data and revised the manuscript.   The authors would like to thank the staff, students, parents and board of directors at Aldrich 4 2 3

Contributions of authors
Memorial Nursery School for their support in allowing us to conduct this study. We for wrapping of wooden blocks and markers with paper and cleaning up these objects after Mayo Clinic Cancer Center, for use of the xCELLigence MP equipment for the infectivity assay.