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Molecular and Microscopic Analysis of Bacteria and Viruses in Exhaled Breath Collected Using a Simple Impaction and Condensing Method

Posted on February 27, 2020 at 7:35 AM

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 µL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses.


Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 µL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m3 in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5–1.0 µm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix.



Bioaerosols are present virtually anywhere in the environment, and their exposure is shown to cause numerous adverse health effects [1]–[2]. In addition, there is also a possible release of bio-warfare agents in a man-made bio-terror event. A number of studies demonstrated that the respiratory tract can be colonized with disease organisms [3]–[5]. Through talking, coughing, sneezing or singing, the potential virulent organisms can be exhaled and spread into the ambient environment [6], which accordingly causes air contamination. For example, SARS in 2003 and H1N1 in 2009 outbreaks were shown to be attributed to the airborne route of disease transmission [7]–[10].


Among many other diseases, respiratory infection accounts for 23.3–42.1% of the total hospital infections [11], and is listed as the third leading killer [12]. However, present diagnosis procedures using nasal swabs, bronchoalveolar lavages, nasopharyngeal aspirates or sputum samples, appear to cause unpleasant experiences in addition to long detection time. During flu outbreaks, body temperature or isolation procedures are often used to control and prevent further spread, however such methods are lacking scientific evidence and not always effective with those patients infected but in latent period. On another front, exhaled breath condensate (EBC) as a simple and noninvasive method is increasingly being utilized in early disease screening and infectious aerosols measurements, e.g., lung cancer [13], [14], asthma [15], [16], and other respiratory problems [17], [18]. In previous studies, human influenza A viruses were detected in exhaled breath using EBC [19], [20] as well as filter [21], mask [22], [23] and a liquid sampler [24]. In another study, foot-and-mouth disease viruses were also found in the exhaled air from experimentally infected cattle [25]. In addition, high levels of bacterial concentrations in EBC were also observed in other studies [26]–[29]. It was recently shown that exhaled breath could be also analyzed for fungal infection by relevant biomarker, e.g., 2-Pentyl furan (2PF) for aspergillosis [30]. Overall, EBC has demonstrated great potential and advantages in early disease screening and diagnosis [31], opening a new arena for studying airway inflammation and chemistry [32]. Recently, Vereb et al (2011) suggested that exhaled breath can be also used for assessing a variety of environmental exposures. .....

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