All human studies (Fig. 5) at our three study sites (Bangalore, Marburg, Boston) were conducted in accordance with relevant regulations, and in accordance with the Declaration of Helsinki. Exclusion criteria included at all three sites included: human volunteers younger than 18 years or older than 70; negative (for Germany and US) or positive (India) nasopharyngeal swab for SARS-Cov-2 (RT-PCR) (within 1 week); subjects with chronic debilitating illness like cancer, immune deficiency etc.; and notably in at Bangalore Baptist Hospital severely ill subjects, subjects with mental illness/psychiatric medications, and subjects unable to complete 3 full days of treatment with active intervention or placebo.
Figure 5 Flowchart of multisite human volunteer study. Full size image
Germany study site: exhaled aerosol study recruitment and enrollment
We recruited 357 healthy human subject volunteers at the Klinik Sonnenblick, Marburg from January to March 2021. Of these there were 212 male and 145 female, 65 smokers and 292 non-smokers, ages 18–83, and BMI 17 to 43 (see Supplemental Material). We obtained IRB approval for the study from the Ethics Committee at the University Marburg. All participants provided written informed consent prior to enrollment. Age, weight, height, and smoking status was documented for all of the subjects and lung health parameters documented for a subset of 157 subjects (see Supplemental Material). Subjects exhaled aerosol at normal tidal breathing and their exhaled aerosol particle numbers were assessed as further described below.
India study site: COVID-19 patient study recruitment and enrollment
We recruited 87 human subject volunteers ages 16–57 among mildly symptomatic COVID-19 patients at Bangalore Baptist Hospital (BBH) during a phase of the Indian pandemic (December to June 2021) over which sequenced BBH infections of the delta (B.1.617.2) coronavirus variant increased from a small minority of cases to greater than 60% of Indian infections40. Participants were screened for SARS CoV-2 infection by polymerase chain reaction (PCR) before enrollment. We obtained IRB approval for the study from the Ethics Committee at Bangalore Baptist Hospital. All participants provided written informed consent prior to enrollment. We assessed symptoms of all 87 participants immediately after enrollment by analysis of blood markers of inflammation (CRP, D-Dimer), lung X-Ray, pulse oximetry, temperature and self-reported symptoms (fever, cough, diarrhea, loss of taste/smell, breathing difficulty, body pain). Exhaled particles of all participants were measured by the particle detector method described below, and 40 were randomly assigned to two treatment cohorts (Table 1) with a block randomization design in which patients, clinical and research staff were all blinded to the use of the active or the control. We used a sequentially numbered, opaque, sealed envelope (SNOSE) technique in which the randomization group is written on a paper kept in an opaque sealed envelope, which is labeled serially. For each person recruited, the numbered envelope corresponding to that person's recruitment number is opened to see the allotment category. An active (FEND) cohort of 20 subjects received by nasal inhalation a calcium-rich hypertonic salt solution (4.72% CaCl 2 , 0.31% NaCl) via a hand-held, vibrating-mesh nebulizer (see Supplemental Material) and with a median volume droplet diameter of 8–12 μm designed to target the nose, trachea and main bronchi. A (Simply Saline) control cohort of 20 subjects received by nasal spray an isotonic saline (0.9% sodium chloride) with droplets sized to deposit in the nose of approximately 50 μm mean diameter. All participants received the active or the control three times a day over the first three days following the start of the study. Patients were blinded to whether they received the active or the control. We evaluated exhaled aerosol of all active and control patients before and after salt administration for one to two hours post-delivery. We monitored all patients each day for oxygen saturation levels, body temperature, IV antibiotic and steroid treatment (where needed), and self-reported symptoms on a scale of 1–5 (increasing in scale from no symptoms to the most severe symptoms). We used face-mask sampling to detect and quantify exhaled SARS-CoV-2 as further described in the Supplemental Material. We measured C-Reactive Protein (CRP) and D-Dimer in blood samples of all participants at the commencement of the study to assess inflammation and need for intravenous antibiotics. On the first day and each subsequent day we measured oxygen saturation by pulse oximetry. All participants in the India study received oral antibiotics (azithromycin) and were treated for fever with paracetamol. We responded to persistent fever or bad cough not relieved by symptomatic management by administering intravenous antibiotics. We administered steroid in response to persistent distressing symptoms, falling oxygen saturation (below 95%) or increased CRP or D Dimer. Failure to maintain adequate oxygen saturation levels or reduce distressing symptoms resulted in escalation to intensive care. We excluded results from the randomized control study of all subjects who escalated to intensive care before completing the three days of FEND or Simply Saline administration. We also excluded from the study those patients who required supplemental oxygen prior to admission or during the first three days post admission (blood oxygen saturation below 95%).
US study site: exercise-induced dehydration study recruitment and enrollment
We recruited at the R3VIVE Fitness in Boston Massachusetts 20 healthy human subject volunteers, 13 male and 7 female, no smokers, ages 22–37, and BMI 22 to 33 (see Supplemental Material). Participants were randomly assigned to treatment and non-treatment groups by choosing between blank envelopes in which their identity as Active or Control was identified. Each participant exercised in two phases of 30 min each. After the first 30 min participants exhaled aerosol was measured a second time. Those participants in the active group received by nasal inhalation the calcium-rich hypertonic salt solution via a second hand-held, mechanical-pump spray and with a median volume droplet diameter of 8–12 μm (Supplemental Material). At the end of the 60 min of exercise the exhaled aerosol of all participants was assessed a final time. Body weight was measured for all of the subjects at multiple intervals without change of clothing. Body weight was measured using a using an InBody H20N Smart Full Body Composition Analyzer Scale with an accuracy of 0.2 lbs. Oxygen saturation was measured for several of the subjects at the t = 0, 30 min and 60 min, and several of the subjects also exhaled aerosol at t = 0 with a residual volume breathing maneuver. Pulse oxygen saturation pressure was measured using a Massimo pulse oximeter (Mighty Sat). We obtained an exemption from IRB approval from E&I Review. All participants provided written informed consent prior to enrollment. Six subjects repeated maneuvers on two separate days, and 4 subjects were excluded from the study on one of the two days of the study either for having consumed water during the study or starting a breathing maneuver without allowing time to recover normal tidal breathing—forced (residual-volume) breathing leading to an amplification of respiratory droplet generation as further discussed in the Supplemental Material.
Exhaled aerosol assessment
We measured exhaled particles in two independent ways in our three studies in the US, Germany and India. In the US and India studies we used a Non-Dried Droplet Counter Method—which has the advantage of assessing the actual droplet sizes as exhaled while the disadvantage of exhaled droplet size depending on atmospheric conditions. Exhaled particles were measured before and after administration of the active or the control by a particle detector (Climet 450-t) designed to count airborne particles in the size range of 0.3 μm to greater than 5 μm. The particle detector air port was attached by a flexible plastic tube to the side (by a T connector) of a 1″ inner diameter tube into which subjects inhaled and exhaled. The 1″ tube connected at one end a mouthpiece provided with standard nebulizer tubing and at the other end a portable HEPA filter. The entire tubing system facilitated the filtration of all environmental particles from the lungs of subjects over a period of about one minute of breathing with subjects’ lips tightly sealed around the mouthpiece and pinching their noses. The rate of flow of the particle counter (50 L/min) was near the typical peak inspiratory/expiratory rate of flow of human subject breathing such that the direction of air flow remained into the particle counter. Each standard nebulizer tubing and mouthpiece were removed from sealed packaging before each subject prior to the subject’s first exhaled particle detection. On subsequent counting procedures, the same mouthpiece, tubing and HEPA filter were reattached by the participant to insure the absence of contamination from one subject to the next. Before each test, the mouthpiece was replaced by a stopper and the particle detector was turned on to verify the absence of leakage of particles from the environment. Background of less than 10 particles per liter of air was deemed “well sealed”. With the mouthpiece placed back onto the tubing, subjects performed normal tidal breathing through the mouthpiece while plugging their noses with their fingers over 1 to 2 min—beginning with two deep breaths to empty their lungs of environmental particles. Over this time frame particle counts per liter of air pulled from the exhaled breath into the particle counter diminished and subsequently fluctuated around a baseline number. Given the assurance of no leakage from the outside environment, the tight lip seal and the pinched nose, we assumed this baseline number to equate to the particles generated within the subject’s airways. Once the lower plateau of particle counts was reached subjects continued to breathe normally for the determination of exhaled aerosol particle number. Participants sat opposite to the study administrator with a plexiglass barrier in between. The Climet 450-t particle counter reports particle counts as a function of aerodynamic particle size ranges for particles larger than 0.3 μm, particles larger than 0.5 μm, particles larger than 1 μm, and all particles larger than 5 μm. The numbers reported represent average values of particle counts automatically measured by the light-scattering detector over six seconds. For our determination of exhaled aerosol particle number we averaged three to eight average particle counts (each integrating a six second interval) as reported by the particle detector to determine the mean exhaled particle count and the standard deviation. In the German studies we used a Dried Droplet Counter Method. This approach has the advantage of controlling humidity variability in the drying of exhaled droplets by shrinking exhaled droplets in the process of counting—while it is an underestimate of the actual droplet sizes exhaled. Exhaled particles are counted and sized in a similar way as in the Non-Dried Method, while involving drying of exhaled droplets and assessment of droplets as small as 150 nm. The Dried Droplet Counter method involved an aerosol spectrometer (Resp-Aer-Meter, Palas GmbH, Karlsruhe, Germany), specifically designed to detect airborne exhaled particles in the size range of 0.15–5.0 μm with very high sizing resolution (16 channels/decade). The optical sensor utilized a polychromatic light source to create a well-defined optical measurement volume, with every particle traveling through the measured volume generating a scattered light pulse. The size and quantity of particles were determined from the number and intensity of the scattered light pulses. Given the lower flow-rate through the system relative to the US and India measurement system (for the German system the flow rate was 9.5 L/min), the instrument comprised a heated hose section upstream of the measurement cell to avoid condensation effects and enable evaporation of larger droplets. The temperature and relative humidity in the sampled air was measured. Exhaled breath from subjects was collected as with the Non-Dried Droplet Counter by a t-adapter with HEPA filter, mouthpiece and connection port to the Resp-Aer-Meter via a hose. Again, to ensure effective hygiene, each sampling kit was removed from sterile packaging before each exhalation maneuver was initiated. Patients and healthy controls performed quiet tidal breathing through the mouthpiece while the nose was closed via a nose clip. The measurement lasting 1–1.5 min to determine the quantity of particles emitted from the lungs. The results of the test were directly displayed as a graphical curve (Fig. 1C), enabling calculation of the mean exhaled particle count per liter, particle size distribution, and mean particle size (in µm).
Airway hydration delivery
We delivered the hypertonic calcium-rich salts to the upper airways with two different hand-held aerosol generators in our US and India studies. Both (see Supplemental Material) yielded salt mist droplets with a median diameter in the 8–12 μm range. Each delivered per 4 s actuation approximately 25 mg of hypertonic salt solution. Two deep nasal inhalations of approximately 4 s constituted an administration with each of the aerosol generators.
Statistical analysis
All error bars represent 95% confidence intervals based on standard deviation values. Significance of differences in individual and collective aerosol numbers were determined by twin-tailed T Test. We calculated statistical significance of differences using a multiway analysis of variance (ANOVA) test for each set of variables. This allowed for evaluating the influence of multiple factors on the mean within a 95% confidence interval. P-values were calculated for each unique set of variables compared to baseline values. Each P-value below 0.05 was considered to be statistically different.