Molecular and microscopic analysis of bacteria and viruses in exhaled breath collected using a simple impaction and condensing method.

Zhenqiang Xu,Fangxia Shen,Yan Wu,Maosheng Yao,Qi Chen,Xiaoguang Li,Xu Jie,Leo L M Poon

doi:10.1371/journal.pone.0041137

Abstract

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.

Highlights

Bioaerosols are present virtually anywhere in the environment, and their exposure is shown to cause numerous adverse health effects [1,2]
Body temperature or isolation procedures are often used to control and prevent further spread, 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]
The time needed for 100 ml EBC including sample collection and removal was around 2 min

Summary

Introduction

Bioaerosols are present virtually anywhere in the environment, and their exposure is shown to cause numerous adverse health effects [1,2]. Development of exhaled breath condensate collection device and method

Results

Conclusion