Rapid detection of Azole resistance genotypes of A. fumigatus

Yu et al. [1]: A quick summary of interesting points.

Sibbe Bakker
sibbe.bakker@wur.nl

Why is rapid detection important?

  • Current methods rely on qPCR.

  • This can be slow and cumbersome.

  • The Yu et al. suggest a new lab-on-a-chip method.

qPCR overview from QIAGEN [2]

What is the Yu et al. study about?

  • Rapid detection of azole resistance A fumigatus.

  • Lab-on-a-chip technology.

  • Limit of detection: 10 copies.

  • Measurement time1: 30 minutes.

  • Tested on 30 isolates.

How does it work?

Overview

The main innovation of Yu et al. [1]: The lab-on-a-chip assay.

Loop mediated isothermal amplification (LAMP)

How does it work?

Overview of the LAMP assay made by Park et al. [3]

How is the result commonly measured?

  • Agarose gel eletrophoresis [4].
  • Turbidity [5].
  • Fluoresence [4].
  • Endpoint colourmetry [6].

Benifits and drawbacks of the LAMP assay [7]

Benifits

  • The reaction is quicker than PCR.

  • The reaction does not need specialist equipment.

Drawbacks

  • Primer design is complex.
  • Cross contamination can be an issue.
  • Cannot be multiplexed easily.

Lab on a chip

How does it work?

  • When DNA polymerase encorporates nucleotides, protons are released.

  • Increase in pH is measured.

  • Lab-on-a-chip has already known usages for LAMP assays:
    • Detection of Salmonella in food [8].
    • Detection of Covid-19 [9].
  • Cost of lab-on-a-chip [10]:
    • Research settings are €60 for a chip (€3 per reaction).
    • Can be mass-produced for €0.60 per reaction.

Outlook

How does it compare to the gold standard?

Relation between qLAMP and eLAMP for the time to positive value for the 20 A. fumigatus isolates.

  • The work of Yu et al. [1] has been not been followed yet:

    • Human samples have not been directly analysed.
    • Development of a ‘sample-to-answer’ device.

  • Is this useful work for future environmental sampling work?

  • How would this method need to be improved?

Questions?

Let’s discuss this paper!

Extra slides

Justification of sample size

  • Sample size estimation
    \(n\geq1.96^2 p (1-p) / x^2\), \(x\) is margin of error, \(p\) is suspected sensitivity.

  • ((1.96^2) * 0.95 * 0.05) / (0.1^2) = 18.2476

  • rounded up to 20 isolates.

Genetic diversity of isolates

Maximum likelyhood phylogeny of A. fumigatus isolates used in the [1]. `

References

Fungal Genetics Meeting

1.
Yu L-S, Rodriguez-Manzano J, Moser N, et al (2020) Rapid Detection of Azole-Resistant Aspergillus fumigatus in Clinical and Environmental Isolates by Use of a Lab-on-a-Chip Diagnostic System. Journal of Clinical Microbiology 58(11):10.1128/jcm.00843–20. https://doi.org/10.1128/jcm.00843-20
2.
What is Real-Time PCR (qPCR)? | Bio-Rad. https://www.bio-rad.com/en-nl/applications-technologies/what-real-time-pcr-qpcr?ID=LUSO4W8UU. Accessed 11 Dec 2023
3.
Park J-W (2022) Principles and Applications of Loop-Mediated Isothermal Amplification to Point-of-Care Tests. Biosensors 12(10, 10):857. https://doi.org/10.3390/bios12100857
4.
Shirato K (2019) Detecting amplicons of loop‐mediated isothermal amplification. Microbiol Immunol 63(10):407–412. https://doi.org/10.1111/1348-0421.12734
5.
Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289(1):150–154. https://doi.org/10.1006/bbrc.2001.5921
6.
Goto M, Honda E, Ogura A, Nomoto A, Hanaki K-I (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. BioTechniques 46(3):167–172. https://doi.org/10.2144/000113072
7.
Wong Y‐P, Othman S, Lau Y‐L, Radu S, Chee H‐Y (2018) Loop‐mediated isothermal amplification (LAMP): A versatile technique for detection of micro‐organisms. Journal of Applied Microbiology 124(3):626–643. https://doi.org/10.1111/jam.13647
8.
Sun Y, Linh Quyen T, Quang Hung T, Hoe Chin W, Wolff A, Duong Bang D (2015) A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. In food samples. Lab on a Chip 15(8):1898–1904. https://doi.org/10.1039/C4LC01459F
9.
Rodriguez-Mateos P, Ngamsom B, Walter C, et al (2021) A lab-on-a-chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resource-limited settings. Analytica Chimica Acta 1177:338758. https://doi.org/10.1016/j.aca.2021.338758
10.
Will McManus “Lab on a chip” genetic test device can identify viruses within three minutes with highest accuracy. https://www.bath.ac.uk/announcements/lab-on-a-chip-genetic-test-device-can-identify-viruses-within-three-minutes-with-highest-accuracy/. Accessed 11 Dec 2023
11.
Sewell TR, Zhu J, Rhodes J, et al (2019) Nonrandom Distribution of Azole Resistance across the Global Population of Aspergillus fumigatus. mBio 10(3):e00392–19. https://doi.org/10.1128/mBio.00392-19

Footnotes

  1. Measurement time is excludes the DNA extraction step.