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Diagnostics to reduce antimicrobial (mis)-use

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

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Diagnostics to reduce antimicrobial (mis)-use

  1. 1. Diagnostics to reduce antimicrobial (mis)-use Elise Schieck, Caroline Fossum, Bernt Hjertner SLU, Uppsala, 6 December 2019 Viktor Ahlberg
  2. 2. The idea Development of a test that discriminates between bacterial and viral infections
  3. 3. The idea – Why? to treat not to treatOR
  4. 4. The idea – Why? to treat not to treatOR Hypothesis: Proper diagnosis will reduce antimicrobial (mis)-use.
  5. 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. 6. Background Sligthly modified from: Mittal, R et al. 2014. Int J. Infectious Diseases 29: 259-267 Overlap between bacterial and viral pathways
  7. 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. 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. 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. 10. Primer design
  11. 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. 12. Testing shortlisted primers - qPCR Shortlisted genes: Ref genes: SDHA YWHAZ ”Viral” genes: MxA IFI44L RSAD2 “Bacterial” genes FAM89A SLP1 S100PBP YWHAZ FAM89A
  13. 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. 14. Testing on reference and clinical samples Refgenes Viral Bacterial 2216/polyIC PMA/ionomycin
  15. 15. Testing on reference and clinical samples Refgenes Viral Bacterial Haemophilus day 0 Haemophilus day 4 Actinobacillus acute
  16. 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. 17. Conclusion Development of a test that discriminates between bacterial and viral infections - Is it possible?
  18. 18. Conclusion Development of a test that discriminates between bacterial and viral infections - Is it possible? - Yes it is!
  19. 19. Conclusion Development of a test that discriminates between bacterial and viral infections - Is it possible? - Yes it is!
  20. 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. 21. Thank you! Ulf Magnusson and Vish Nene Benjamin Nzau
  22. 22. This presentation is licensed for use under the Creative Commons Attribution 4.0 International Licence. better lives through livestock ilri.org ILRI thanks all donors and organizations which globally support its work through their contributions to the CGIAR Trust Fund

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