Presented by Elise Schieck, Caroline Fossum and Bernt Hjertner at Swedish University of Agricultural Sciences, 6 December 2019

1. Diagnostics to reduce antimicrobial (mis)-use
Elise Schieck, Caroline Fossum, Bernt Hjertner
SLU, Uppsala, 6 December 2019
Viktor Ahlberg

2. The idea
Development of a test that discriminates
between bacterial and viral infections

3. The idea – Why?
to treat not to treatOR

4. The idea – Why?
to treat not to treatOR
Hypothesis:
Proper diagnosis will
reduce antimicrobial
(mis)-use.

5. The idea – How?
Development of a test that discriminates
between bacterial and viral infections
How ?
Identify host responses typical
for bacterial versus viral infections

6. Background
Sligthly modified from: Mittal, R et al. 2014. Int J. Infectious Diseases 29: 259-267
Overlap between bacterial and viral pathways

7. Background
Discovery group: 240 children
52 definite bacterial infection,
92 definite viral infection,
96 indeterminate infection.
Validation group: 130 children
23 definite bacterial,
28 definite viral,
79 indeterminate infections
Identification of bacterial infections:
90-100% sensitivity
94-96% specificity

8. Selection of candidate genes
Gene Name Definition
Definite Bacterial vs.
Definite Viral
log2 FC q-value
Upregulated in viral infections
IFI27 interferon alpha inducible protein 27 -4.32 4.78E-21
IFI44L Homo sapiens interferon-induced protein 44-like, mRNA. -3.79 1.36E-22
IFIT1 interferon induced protein with tetratricopeptide repeats 1 -3.49 2.90E-20
RSAD2 radical S-adenosyl methionine domain containing 2 -3.28 5.14E-19
IFIT3 interferon induced protein with tetratricopeptide repeats 3 -2.66 2.47E-16
OTOF otoferlin -2.53 1.73E-14
MxA MX dynamin like GTPase 1
Upregulated in bacterial infections
FAM89A family with sequence similarity 89 member A 1.21 2.97E-14
UPB1 beta-ureidopropionase 1 1.23 5.27E-13
PTPN20 protein tyrosine phosphatase non-receptor type 20 1.35 1.88E-11
TMEM119 transmembrane protein 119 1.35 1.76E-08
SLPI secretory leukocyte peptidase inhibitor 1.84 2.44E-12
S100P S100 calcium binding protein P 2.35 1.39E-16
PI3 peptidase inhibitor 3; elafin precursor 2.58 4.52E-10
CRP C-reactive protein

9. Primer design
• Orthologues for selected genes were identified in S.
scrofa by sequence homology.
• 6 reference genes were identified from Uddin et al.
(2011) and new primer pairs designed for them.
• Primers were designed using NCBI’s Primer-BLAST
tool and their specificity confirmed using BLASTN.

10. Primer design

11. Testing Primers – conventional PCR
Reference genes
“Viral” genes “Bacterial” genes
# Primer ID BP
1 FAM89A 1 97
2 FAM89A 5 142
3 UBP1 1 102
4 UBP1 2 101
5 PTPN20 2 149
6 PTPN20 10 83
7 TMEM119 1 108
8 TMEM119 4 80
9 SLP1 1 113
10 SLP1 7 94
11 S100PBP 4 136
12 CRP 4 75
13 PI3 1 81
# Primer ID BP
1 MxA 1 99
2 MxA 4 109
3 IFI27 2 140
4 IFI27 3 102
5 IFI44L 1 104
6 IFI44L 8 177
7 IFIT1 N 141
8 RSAD2 4 102
9 RSAD2 8 123
10 IFIT3 6 136
11 OTOF 1 97
12 OTOF 7 104
# Primer ID BP
1 RPL4 udding 185
2 RPFL 107
3 TBP udding 118
4 TBP 135
5 B2M udding 180
6 B2M 88
7 PPIA udding 171
8 PPIA 109
9 SDHA udding 149
10 SDHA 113
11 YWHAZ udding 146
12 YWHAZ 114

12. Testing shortlisted primers - qPCR
Shortlisted genes:
Ref genes:
SDHA
YWHAZ
”Viral” genes:
MxA
IFI44L
RSAD2
“Bacterial” genes
FAM89A
SLP1
S100PBP
YWHAZ
FAM89A

13. Testing shortlisted primers - qPCR
Shortlisted genes:
Ref genes:
SDHA
YWHAZ
”Viral” genes:
MxA
IFI44L
RSAD2
“Bacterial” genes
FAM89A
SLP1
S100PBP
YWHAZ
FAM89A
PPIA

14. Testing on reference and clinical samples
Refgenes
Viral
Bacterial
2216/polyIC PMA/ionomycin

15. Testing on reference and clinical samples
Refgenes
Viral
Bacterial
Haemophilus day 0 Haemophilus day 4
Actinobacillus acute

16. Testing on reference and clinical samples
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
LF524 SH86 pig No 14 day 4 Actbac pig 25
Average viral vs bacterial
viral bacterial

17. Conclusion
Development of a test that discriminates
between bacterial and viral infections
- Is it possible?

18. Conclusion
Development of a test that discriminates
between bacterial and viral infections
- Is it possible?
- Yes it is!

19. Conclusion
Development of a test that discriminates
between bacterial and viral infections
- Is it possible?
- Yes it is!

20. Next steps
• Improve/ test further bacterial biomarkers
• Optimization of assays
• Test on more clinical/trial samples (viral and bacterial
infections
• Develop similar assays for other livestock (cattle and goats)
• Identify suitable technology for the final test

21. Thank you!
Ulf Magnusson and Vish Nene
Benjamin Nzau

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