A mobile biosafety microanalysis system for infectious agents

Beniac, Daniel R.; Hiebert, Shannon L; Siemens, Christine G.; Corbett, Cindi R.; Booth, Tim F.

doi:10.1038/srep09505

Cited by 6 publications

(9 citation statements)

References 16 publications

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“…25 Recently, rapid methods of sample preparation and transportable EM equipment, which makes EM an easier and faster tool to use, have also been reported to improve the identification of infectious agents. [26][27][28] These studies and our data collectively support that in future, TEM may be used along with other diagnostic tools, as one part of a comprehensive assay system for microbiological diagnosis of severe pneumonia. Several imitations should be noted in the present study.…”

Section: Discussionsupporting

confidence: 79%

Transmission Electron Microscopy Improves the Diagnostic Sensitivity in Nonbacterial Etiology of Severe Pneumonia: A Retrospective Study

José

et al. 2019

The American Journal of the Medical Sciences

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Background: Severe pneumonia is responsible for great mortality and morbidity worldwide, and early-applied effective antiinfective therapy can improve the prognosis of patients. However, identification of infectious agents in severe pneumonia remains a major challenge so far. In this study, the potential utility of transmission electron microscopy (TEM) in detecting nonbacterial pathogens in patients with severe pneumonia was retrospectively evaluated.Materials and Methods: A total of 106 patients diagnosed with severe pneumonia at our hospital from September 2015 to December 2017 were included, and their baseline clinical characteristics were collected. Nonbacterial infectious agents detected by TEM in bronchoalveolar lavage fluid (BALF) and serological tests were summarized. The detection rates were further compared between TEM and serological tests. Results: BALF examination under the transmission electron microscope revealed 24 viruses, 16 mycoplasmas, 18 chlamydia, 2 fungi and 74 bacteria in 99 samples, among which 61 samples were mixed infections. The combined use of serological tests and TEM significantly improved the detection rate of nonbacterial infectious agents in patients with severe pneumonia.Conclusions: Our data support that implementation of TEM could improve the sensitivity for detecting viruses, atypical pathogens and mixed infections in BALF from patient of severe pneumonia. Therefore, TEM may be used as an auxiliary diagnostic method of other microbiological tests in severe pneumonia.

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Section: Discussionsupporting

confidence: 79%

Transmission Electron Microscopy Improves the Diagnostic Sensitivity in Nonbacterial Etiology of Severe Pneumonia: A Retrospective Study

José

et al. 2019

The American Journal of the Medical Sciences

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“…In the past, the difficulty with specimen preparation methods for SEM analysis was the major cause of its limited use in the routine analysis of microbiology 32,33 . Two other problems were also counted for achieving high-resolution SEM images of microbes: nonconducting behavior and the presence of moisture on microbial samples.…”

mentioning

confidence: 99%

“…Two other problems were also counted for achieving high-resolution SEM images of microbes: nonconducting behavior and the presence of moisture on microbial samples. Both of these reasons decrease the performance of the microscope, which reduces contrast and resolution 32,33 . Due to the drying problem, specimens become collapsed, shrunken and distorted, even after chemical fixation.…”

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confidence: 99%

Power of Scanning Electron Microscopy and Energy Dispersive X-Ray Analysis in Rapid Microbial Detection and Identification at the Single Cell Level

Khan

Kim

2020

Sci Rep

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The demand for rapid, consistent and easy-to-use techniques for detecting and identifying pathogens in various areas, such as clinical diagnosis, the pharmaceutical industry, environmental science and food inspection, is very important. In this study, the reference strains of six food-borne pathogens, namely, Escherichia coli 0157: H7 ATCC 43890, Cronobacter sakazakii ATCC 29004, Salmonella Typhimurium ATCC 43971, Staphylococcus aureus KCCM 40050, Bacillus subtilis ATCC 14579, and Listeria monocytogenes ATCC 19115, were chosen for scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. In our study, the time-consuming sample preparation step for the microbial analysis under SEM was avoided, which makes this detection process notably rapid. Samples were loaded onto a 0.01-µm-thick silver (Ag) foil surface to avoid any charging effect. Two different excitation voltages, 10 kV and 5 kV, were used to determine the elemental information. Information obtained from SEM-EDX can distinguish individual single cells and detect viable and nonviable microorganisms. This work demonstrates that the combination of morphological and elemental information obtained from SEM-EDX analysis with the help of principal component analysis (PCA) enables the rapid identification of single microbial cells without following time-consuming microbiological cultivation methods. Rapidly detecting and identifying biological threat microorganisms without traditional culture or chemical-based methods are highly important. The widely used identification techniques are nucleic acid-based, biosensor-based and immunologically based techniques. Real-time PCR multiplex PCR, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), and oligonucleotide DNA microarray are examples of some common nucleic acid-based identification techniques 1-4. These techniques have higher sensitivity, specificity, and reliability and can detect multiple pathogens in an automated manner with several constraints, such as sensitivity to PCR inhibitors and complicated primer design, and the methods cannot differentiate viable and nonviable cells 5,6. All of these nucleic acid-based techniques are slow processes that require 4-72 h to detect microbes 1-6. Electrochemical, optical and mass-based biosensors are commonly used to detect microbes. These automated, label-free, real-time detection processes can handle a large number of samples. Biosensor-based processes have several drawbacks, such as long incubation time, numerous washing steps, low specificity, interference with the food matrix and unsuitability for lesser cells 7-10. The lateral flow immunoassay and enzyme-linked immunosorbent assay (ELISA) are two immunological-based detection techniques with several advantages and disadvantages; most importantly, these techniques are also slow processes that require 3-10 h 11,12. These limitations increase the overall cost of the detection process due to costly logistics trails and restrict autonomous operation 13,14. ...

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“…The scanning electron microscope (SEM) can also be useful to reveal morphological features of isolated organisms as well as for diagnosis, but difficulty with specimen preparation methods have in the past limited the use of SEM for routine microbiology 7 8 . Nowadays extremely high quality polycarbonate filters are available: the optimum pore size can be selected to collect any virus or bacterial species (the pores can be as small as 10 nm, less than the smallest viruses).…”

mentioning

confidence: 99%

“…Ionic liquid techniques can also be used safely with infectious pathogens, in a biologically contained SEM enclosure, allowing the characterisation of novel infectious agents in a condition closer to their hydrated “native state” than by conventional sample preparation techniques 8 . In the case of this investigation our conventional protocol involved dehydration in ethanol series, followed by air drying and metal coating before SEM imaging.…”

mentioning

confidence: 99%

The scanning electron microscope in microbiology and diagnosis of infectious disease

Golding

Lamboo

Beniac

et al. 2016

Sci Rep

Self Cite

187

127

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Despite being an excellent tool for investigating ultrastructure, scanning electron microscopy (SEM) is less frequently used than transmission electron microscopy for microbes such as viruses or bacteria. Here we describe rapid methods that allow SEM imaging of fully hydrated, unfixed microbes without using conventional sample preparation methods. We demonstrate improved ultrastructural preservation, with greatly reduced dehydration and shrinkage, for specimens including bacteria and viruses such as Ebola virus using infiltration with ionic liquid on conducting filter substrates for SEM.

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A mobile biosafety microanalysis system for infectious agents

Cited by 6 publications

References 16 publications

Transmission Electron Microscopy Improves the Diagnostic Sensitivity in Nonbacterial Etiology of Severe Pneumonia: A Retrospective Study

Transmission Electron Microscopy Improves the Diagnostic Sensitivity in Nonbacterial Etiology of Severe Pneumonia: A Retrospective Study

Power of Scanning Electron Microscopy and Energy Dispersive X-Ray Analysis in Rapid Microbial Detection and Identification at the Single Cell Level

The scanning electron microscope in microbiology and diagnosis of infectious disease

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