Review 1: "Airborne Pathogen Detection in Fine Aerosol Exhaled Breath Condensates"

RR:ID Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: 

As described in this manuscript, Henderson and his colleagues developed a hand-held exhaled breath condensate collector (the prototype and the commercialized product PBM-HALE) which trapped the saliva prior to the condensation of the exhaled breath, and evaluated its performance in collecting EBC samples from healthy volunteers as well as COVID-19 patients. The developed EBC collector was shown to obtain EBC samples free of salivary contamination, which is supported by the absence of salivary α- amylase in the EBC samples. By successfully separating saliva from the EBC, this study demonstrated different microbiota compositions in the EBC and trapped saliva samples. This study also found that COVID-19 patients yield high levels of SARS-CoV-2 RNA (Ct ± sd: 22.5 ± 2.75) in the EBC samples from forced expiration but not in the samples from tidal breath.

Exhaled breath has increasingly been studied as a non-invasive method for assessing various biomarkers in medical and environmental health research. In particular, in the global efforts to combat the COVID-19 pandemic, breath analysis has significantly contributed to the development of rapid infection diagnosis/screening methods and the elucidation of the airborne transmission of SARS-CoV-2, etc. However, the exhaled breath condensate (EBC), a type of breath sample that is formed mainly by the condensation process of the over-saturated water vapor in exhaled breath, has been doubted as unable to provide consistent and representative samples due to excessive dilution of the analytes and susceptibility to external contamination during the sampling procedure, hindering its further clinical applications. Overall, this manuscript described a successful design of an EBC collector that can provide EBC samples free of saliva and environment contamination and showed promising reproducibility. However, some of the results and conclusions are of technical concerns and need to be interpreted with caution:

1. The authors described that the developed EBC collector separates large droplets by inertial impaction and then condenses fine aerosols. The size cut-off between large droplets and fine aerosols is described as 10 μm in the introduction. However, no aerosol measurement data are available to support this description. According to the salivary α- amylase results, the so-called large droplets contain a substantial fraction of saliva. Therefore, the results only demonstrated the separation of saliva from the EBC sample.

2. Different condensation temperatures have been tested, and the optimal condensation temperature is determined as -78.5 ℃ provided by the dry ice. However, results showed that most of the culturability of microbes and virus infectivity was lost at this condensation temperature, which limits the application of the EBC sample since the culture is still a critical method of preference for infection diagnosis. Therefore, the condensation temperature should be further optimized.

3. The authors state that exhaled particle swell in size and be captured on the condensation surface due to gravitational sedimentation and inertial impaction based on the computation flow modeling and particle measurement results. However, the computation flow modeling did not include any aerosol dynamics, and particles were only measured at the outlet of the collector and compared between different condensation temperatures, which did not provide any information to support the gravitational sedimentation and inertial impaction of particles.

4. Some errors or inconsistencies in the data interpretation are potentially misleading, including stating the EBC collection curve is linear in the logarithmic coordinates (Fig 2F), that the results are not from adequate statistical replicates (Fig 1), describing the sample collection duration using mixed measures (e.g., breath counts in Fig 1B, 3C-E, 5A; and collection time in Fig 2F, 3B, 6B-D).