Presented by Arshnee Moodley and Elise Schieck, ILRI, 1 December 2020

1. Diagnostic process
Arshnee Moodley and Elise Schieck, ILRI
1 December 2020

3. A short review of rapid nucleic acid based diagnostic technologies
Background:
Development of a test that discriminates between bacterial and
viral infections -using biomarkers
to treat not to treat
OR

4. A short review of rapid nucleic acid based diagnostic technologies
Background:
Development of a test that discriminates between bacterial and
viral infections -using biomarkers
ILRI and SLU have teamed up to test the possibility of developing an easy-to-use and
cheap diagnostic test that can differentiate between bacterial and viral infections.
Bacterial and viral infections typically induce slightly different responses in the hosts,
and we are currently testing the possibility of using these host markers to develop a
quick and easy-to-use field test.
This may reduce the use of antibiotics to animals infected with viruses.

5. A short review of rapid nucleic acid based diagnostic technologies
Diagnostics of infectious diseases
Pathogen specific
Direct identification
• Culturing
• Antigen testing (ELISA etc)
• DNA or RNA
Indirect identification
• Antibody (host) response (ELISA etc)
Pathogen non-specific
• Clinical symptoms
• Blood count
• CRP
Lab based Point of care (POC) test
Qualitative Quantitative

6. A short review of rapid nucleic acid based diagnostic technologies
Diagnostics of infectious diseases
Pathogen specific
Direct identification
• Culturing
• Antigen testing (ELISA etc)
• DNA or RNA
Indirect identification
• Antibody (host) response (ELISA etc)
Pathogen non-specific
• Clinical symptoms
• Blood count
• CRP
Lab based Point of care (POC) test
Qualitative Quantitative
Out biomarker:
the host’s
transcriptional
profile
• RNA based
• Quantitative
• POC

7. A short review of rapid nucleic acid based diagnostic technologies
Point-of-Care Diagnostics
-Lateral Flow Assays (LFA)
Trends Biotechnol . 2011 May ; 29(5): 240–250. doi:10.1016/j.tibtech.2011.01.007.

8. A short review of rapid nucleic acid based diagnostic technologies
Gootenberg et al., Science 356, 438–442 (2017)

9. A short review of rapid nucleic acid based diagnostic technologies
DNA (or RNA) amplification
Trends Biotechnol . 2011 May ; 29(5): 240–250. doi:10.1016/j.tibtech.2011.01.007.

10. A short review of rapid nucleic acid based diagnostic technologies
Point-of-Care Diagnostics on the market

11. A short review of rapid nucleic acid based diagnostic technologies
From presentation by Anne Liljander

12. A short review of rapid nucleic acid based diagnostic technologies

13. A short review of rapid nucleic acid based diagnostic technologies

14. A short review of rapid nucleic acid based diagnostic technologies

15. A short review of rapid nucleic acid based diagnostic technologies
But….
Used RNA isolation kits
Then NASBA
Then paper based detection,
in 96 well plates

16. A short review of rapid nucleic acid based diagnostic technologies
Summary
Rapidly evolving field
Loads of new possibilities
Watch this space…

20. But, If the plate is pure and you are confident- proceed to
identification

24. Traditional tests: 2 days to get to atleast Genus level

25. Maldi-TOF MS
• Matrix-assisted laser
desorption/ionization time-of-
flight mass spectrometry
• Method adpated to identify
bacterial species based on their
protein profile
• Sample (colony material) is
placed on a slide and overlaid
with a matrix, which dissolves
the cell wall and binds released
material (mainly proteins)

26. The matrix absorbs the laser light
and vaporizes, along with the
sample, and in the process gaining
gains an electrical charge
(ionization)
Electrical fields then guide the ions into
the time of flight mass spectrometer, ions
are separated according to their mass to
charge ratio, and ultimately the quantity
of each ion is measured

27. •Comparison to reference spectra in a
database representing well characterized
bacteria is used to determine the species
Dias 27

28. BD Bruker MALDI Biotyper®
Microflex LT/SH IVD
(8604743)
BD Bruker MALDI
Biotyper® Smart IVD
(8605200)
51 cm x 68 cm x 110 cm
- 84 kg - <50db
200 samples per hour
53 cm x 68 cm x 110 cm - 99 kg-
<60db
400 samples per hour
Biotyper: Microflex or Smart IVD

29. 8290200
8290190
1839298 8280800
Basic Reagent
48 or 96 spots

30. Different reference databases/modules (Fungi, Mycobacteria, )
Identification

31. Antimicrobial Susceptibility Testing

32. Innoculum: 0.5 McFarland = 1.5 X 108 CFU/ml

33. Innoculate: AIMS dispenser:
Automated 96 well dispenser
• Bottles containing
MHB
• Buy empty bottles
and dosing heads
• Select the dosing
volume and pattern

34. Sensititre panels: Value of MIC
• Minimum inhibitory
concentration is the
concentration
required to visibly
inhibit bacterial
growth
• How
susceptible/resistan
t a strain?
• clinical breakpoints
vs. ECOFFs
Custom plates:

35. Read+ Intrepret: Sensititre™ SWIN™
Epidemiology Module
Epi. Module:
• Database
• monitor and investigate local antibiotic
resistance patterns
• Antimicrobial stewardship

36. How to access these equipment?
• Maldi-TOF MS
• Service only
• Drop of your fresh overnight plates in the morning, log your request with
a budget code and budget holder/PI, result by the afternoon
• Densicheck
• Note: special tubes
• Authorised people
• AIMS automated dispenser
• Special bottles pus dosing heads
• Authorised people
• Sensititre
• Authorised people

37. Acknowledgements

38. This presentation is licensed for use under the Creative Commons Attribution 4.0 International Licence.
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