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A simple novel device for air sampling by electrokinetic capture

Background

Understanding of the microbiology of air, the aerobiome, is an emerging field of discovery. High-throughput sequencing methods are being used to explore the spatiotemporal distribution of bacterial and fungal populations [1–10]. A variety of sampling methods have been used for studying the air microbiome [3, 11–14]. A variety of different sampling devices are currently available to acquire air samples of microbial and viral particles. These technologies include filters, impingers, impactors, and wet or dry cyclones. The underlying principle of impactors, impingers, and cyclones is the use of an abrupt change in direction of airflow so that aerosol particles will continue on to a surface by virtue of their momentum. Filters are microporous membranes, impingers capture on to the surface of a nutrient agar plate for subsequent colony counts, and impactors capture on a solid surface for subsequent elution, as do dry cyclones. Wet cyclones capture by vortexing into a liquid phase. Aerosol particles may also be separated into size classes with multi-stage devices. For existing devices, capture efficiency falls off rapidly with particle size and there is considerable variability in performance [16–18]. All of these devices require pumping against some resistance. Different apparent microbial communities were found from the use of different air sampling techniques [11, 13]. There is thus a need in aerobiome analysis for a sampling procedure that does not bias the measured biodiversity. Here, we introduce the use of a very simple device for collection of samples and show equivalence to a reference method using filtration. In addition, a variety of air sampling methods have been applied to the airborne transmission of disease [19–30].

Brown first described the principle of ionic propulsion in US patents [31, 32]. A corona wire is subject to a high voltage, creating plasma that imparts charge on particles in the vicinity. The charged particles are then propelled by the voltage gradient to electrodes at an opposing potential. The net flow of charged particles imparts forward momentum on the surrounding medium. The result is a net airflow with no moving parts. This principle has been used in commercial air-cleaning devices. Custis et al. used such an air-cleaning device for collecting dust from the air for measuring allergens. We have developed a mini-scale device using the same principle with optimized airflow and an electrode cartridge that is optimized for sample collection (Inspirotec Sampler). We have demonstrated its use for detection of allergens by immunoassay [35, 36] and viruses by quantitative PCR. The device is simple to operate, compact, and can be placed unobtrusively in any environment. Here, we compare performance with a filter reference method for analysis of the aerobiome.

Mold spores

Mold spores are ubiquitous in the environment but vary according to temperature, humidity, season, and other environmental conditions. Table 1 shows the results of 24-h samples in the three locations. The Inspirotec Sampler values have not been corrected for capture efficiency (approximately 20 %, see “Methods” section). If this correction were applied, the values would all be consistent within the standard deviations achievable by the quantitative polymerase chain reaction (qPCR). Those cases where the species was detected by both methods are indicated by green shading. In no instance was a species detectable by the filter and not detectable by the Inspirotec Sampler. In eight instances, the Inspirotec Sampler detected a species that was not detected by the filter, indicated by yellow shading in Table 1. By this criterion, if the filter is considered a gold standard, the sensitivity is 100 % and the specificity is 87 %. However, these are likely to be true positives since the Inspirotec Sampler is sampling a larger volume of air in a given time. To address this concern, we examined the accumulation rate for Eurotium amstelodami on both filters and the Inspirotec Sampler (Fig. 1). This shows that the Inspirotec Sampler processed both a larger volume, and correspondingly, a larger quantity of spore equivalents were captured, when compared to the filter. As with Table 1, these numbers have not been corrected for capture efficiency. It is not clear why the quantity of spore equivalents captured appears to peak at 6 h for both filter sampling and Inspirotec Sampler. Nevertheless, this illustrates the advantage of the high sampling volume of the Inspirotec Sampler. This advantage is compounded by the Inspirotec Sampler’s easier logistical set-up and silent performance.

Bacterial diversity

Timed samples were run in the basement environment with the same schedule as in Fig. 1. Bacterial 16S rRNA amplicon sequencing generated a total of 1,294,310 sequences from 22 samples. When rarified to 9800 sequences per sample, 385,076 operational taxonomic units (OTUs; 97 % identity) were identified. No significant difference in microbial community structure was observed between the Inspirotec Samplers and the reference method with the use of the R Project for Statistical Computing freeware (weighted or unweighted UniFrac distance ADONIS, p > 0.05, R = 0.06). False-discovery rate (FDR) and Bonferroni-corrected p values showed no significant differences in OTU frequencies between platforms. The genus-level community profile generated by both technologies comprised predominantly Acinetobacter, Gordonia, Methylobacterium, and Pseudomonas (Fig. 2). Differences in abundances in Fig. 2 are therefore not significant.

Interestingly, 180 min produced a signal highly similar to the time zero (blank) suggesting that this time frame was insufficient to generate enough biomass for the detection threshold of the amplicon sequencing technology (Fig. 2). However, by 360 min, the community profiles were significantly different from time zero. Reagent-based contamination is known to be an issue and explains the detected signal for blank and 180 min. The significance of the similarity between microbial profiles generated by the filter and Inspirotec Sampler technologies at each time point was assessed using Procrustes analysis including the left and right electrode (technical replicates) of the Inspirotec Sampler as well as the pump-driven filter. Over the course of time, there was no significant difference between either technical replicate or the air filter, despite greater variability between samplers at the starting zero time (Monte Carlo, p > 0.05) (Fig. 3).

Particle size and capture

A prediction of the method of using ionic propulsion to capture the charge particles is that the capture should be independent of particle size of mass, unlike current aerodynamic-based sampling systems. The location of capture is dependent on the force vectors determined by the voltage gradient. Mass may affect particle acceleration and velocity, but the final capture location is determined by a potential well. We therefore explored sample capture with a random sample of air (bathroom of Table 1) and examined the size distribution of captured particles by atomic force microscopy. Particles down to the nanometer range were captured (Fig. 4), as demonstrated using a visual representation of particle size density (Fig. 4a), as well as a size distribution curve, with a significant fraction trailing into the lower range (Fig. 4b).

The Inspirotec Sampler was run in the bathroom for 24-h (Table 1) and scanned (see “Methods” section). The inset shows the lower end of the distribution curve and captured particles extending down into the 500-nm range and below. The sampler is thus able to capture particles going down to very low sizes and in the range that will penetrate the lungs and cause symptoms. This illustrates the range of sizes of not-identified aerosol particles that are captured.

Conclusion

We have demonstrated the applicability of an electrokinetic air sampler for the molecular detection of microorganisms in air. All procedures are of wide applicability to any measurements in the aerobiome. This technology is easily deployed as it can be plugged into any electrical socket, silent, has low visual impact, and so could be readily applied to indoor settings for identifying and tracking emerging pandemics. The device performance was comparable to or exceeded that of the reference method.

Discussion

The device is inexpensive and requires no technical skill to operate, compared with any other competing technology. Both SARS and MERS [39, 40] epidemics may be traced back to human-animal interfaces, and early deployment in such emerging pandemics would facilitate the tracing of early stages and subsequent routes of transmission. In a separate study, we showed the capability of capturing Venezuelan equine encaphilitis virus in a controlled environment chamber at the US Army Edgewood Chemical Biological Center with aerosol particles down to 1 μm. The viruses had been inactivated by gamma irradiation. The result was that a large proportion of the original virions had RNA that was not amplifiable, so the capture efficiency was apparently very low based on the original virus titer. However, analysis by digital PCR using the Poisson distribution at low amplicon concentrations showed that the capture efficiency was in the range of 20–40 % for these articles. Here, the performance exceeded that of the reference method using a microporous filter and showed ability to detect mold spores using EPA-accepted PCR technology. Of the species for which primers and probes were used in the qPCR, Acremonium strictum, Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceus, Aspergillus sydowii, Aspergillus ustus, Aspergillus versicolor, Chaetomium globosum, Cladosporium cladosporioides, Eurotium amstelodami, Memnoniella echinata, Paecilomyces variotii, Penicillium aurantiogriseum, Penicillium brevicompactum, Penicillium chrysogenum (type 2), Penicillium purpurogenum, Penicillium variabile, Scopulariopsis brevicaulis, Stachybotrys chartarum, Trichoderma viride, Ulocladium botrytis, all but Aspergillus ustus, Memnoniella echinata, and Penicillium variabile were detectable as spores over the three locations tested.

Another feature of the electrokinetic propulsion is the ability to capture and measure particles down into the nanoparticle range. Particles generated from respiratory activities in the range of 0.05 to 500 μm are associated with infection. Most commonly used air sampling devices have a cut-off at about 1 μm. Allergens may extend down to a size range that has been missed by current sampling technology, and there is evidence that bacterial endotoxin, which exacerbates the effect of allergens, may exist in size ranges below the size of bacteria. The device described in this publication will have the capability of extending the size range of particles that can be collected from the aerobiome.

Fahlgren et al. found that the diversity of microbial communities captured by different samplers in external environments in Norway and Sweden were similar, whereas Hoisington et al. have found significant inconsistencies between different sampler types at locations within a US retail store. In neither case was consideration made to the time of run needed to resolve reagent background. We showed that a minimum of 6 h sampling was required regardless of the method. We demonstrate the application of ionic propulsion technology to capture a wider range of particle sizes than with traditional air filter sampling, with no significant bias in fungal and bacterial community recovery.

Availability of supporting data

All sequence data will be made available through FigShare, http://dx.doi.org/10.6084/m9.figshare.1603492