1. Dr Ngo Tat Trung, PhD Danang– September 2015
Translating molecular testing into sepsis diagnosis:
the challenge in clinical practices.
Dr Ngo Tat Trung, PhD
(Dept. Molecular biology
108 Military Central Hospital)
Danang– September 2015

2. Sepsis related deaths
Hospitalized sepsis patient
Sepsis related death
Năm
US: patients/year; death/year, death=20-70%; rank 10th; account for 6% death.
European death/ year, rank 3th of death
Harrison et al. Critical Care 2006; Nicasio Mancini, 2010

3. Classical Sepsis detection tools CÁC PHƯƠNG PHÁP CHẨN ĐOÁN NN GÂY NKH
Clinical symptoms
Immune response monitor
Blood culture

4. Blood culture
Classical method, widely implemented but still far from making clinician satisfied because of its intrinsic drawbacks:
Impractical for fastidious pathogens
Time required for first wave of microbial colonies to appear is too long that might switch patients into worse deleterious situation
Large volumes blood is mandatory for proper culturing of aerobic and anaerobic bacteria

5. Consequence of mis/late detection
Risk to be sepsis shock:
Reduce chances to survive
Increased hospital cost
Drug resistance clones emerge
→ need to develop relevent approaches that work complimentary to blood culture
*NicasioMancini et al, 2010

6. PCR combined mass spectrometric DNA sequencing
Nucleic acid test(NAT)

7. Disadvantage:
Supper expensive equipments : including both PCR system and Mass spectrometric installation
Well trained personnel

8. Promising technology, especially for multi-readout assays

9. Challenges to PCR’s sensitivity
An optimized PCR reaction using DNA extracted by Qiagen, Zymo blood extraction kits can only sense pathogen’s ribosomal 16S pieces if the bacterial load exceeds roughly 500 CFU/ml
Patients would present sepsis – related clinical symptoms if bacterial load exceed CFU/ml
Klouche and Schröder 2008

10. How the primers mis-pairing to human DNA happens
NATURE | VOL 431 | 9 SEPTEMBER 2004

11. Two aspects must be focused to harness PCR into sepsis diagnosis
Virut
Two aspects must be focused to
harness PCR into sepsis diagnosis
Human DNA removal/bacterial DNA enrichment
Optimize the conditions for PCR diagnostic algorithm

12. Our strategic resolution
Virut
Our strategic resolution

13. bacterial DNA enrichment
Human DNA removal
bacterial DNA enrichment
Disrupt human blood cells/shear but keep bacterial cells intact
Figure 1: Stepwise pre-analytical protocol to remove human DNA from sepsis- suspected- blood samples
Spin to pellet bacterial cells
Total DNA extraction
Input template for PCR assays

14. Removal of 97-98% of human DNA from blood samples
NaOH- SDS
NaOH- SDS
MCLB1
Beta-globin
6 cycles
Figure 3 Efficiency of human DNA removal: The residues of human DNA after MCLB1 treatment was monitored via beta-globin derived amplification assays: upper panel gel based PCR assay target beta-globin gene; lower panel is sybr green based realtime PCR to quantify residual beta globin gene fragment
Total DNA prepared by NaOH/SDS
DNA processed by MBLC1

15. Bacterial limits of detection Upon human DNA removal
Pseudo sepsis samples (bacterial spiked healthy blood dilution series)
Disrupt human blood cells and shear chromatin by basic pH combined polar detergent /
Spin to pellet bacterial cells
Total DNA extraction
Input template for PCR assays

16. 10000 CFU/ml 1000 CFU/ml Blank control 100 CFU/ml 10 CFU/ml 1 CFU/ml
E. coli
A. baumanii
S.aureus
P. aeruginosa
K. pneumonia
S pneumonia
Figure 4: Sepsis causative pathogens detection limits after human DNA removal
Salmonella
P. mirabilis
S. pidermidis
S. suise
Enterococcus sp

17. Enterobacteriaceae piked healthy blood dilution series

18. Optimize the conditions for PCR diagnostic algorithm

19. Input bacterial DNA extracted after human chromatin removal
Group specfic screening assays 4h
Non-Enterobacteriaceae
Gram(-)
Enterobactericeae
Gram(+) 6h
Figure 2 Group specific – screening algorithm: the first three screening taqman PCR reactions that target bacterial ribosome-16S genes to simultaneously differentiatiate Gram-positive and Gram-negative and Enterobacteriaceae Groups. Samples that are positive in the screening assay will be subjected to genus specific realtime PCR reactions to detect 12 most common sepsis causative pathogens
P. aeruginosa
A. baumannii
N. meningitidis
H. influenza
E. coli
K. pneumoniae
Salmollela
P. mirabilis
Staphylococus sp
Streptococus sp Enterococcocus sp

20. 114 sepsis suspected blood samples recruited
In real clinical diagnosis
Group specific screening realtime PCR analysis
Genus specific realtime PCR confirmation
1.2 ml for In-house human DNA removal
In put DNA
114 sepsis suspected blood samples recruited
Suspected blood sepsis
Figure 5: Study flowchart to compare sepsis causative pathogens diagnostics from direct DNA extraction samples, human DNA removed samples and blood culture.
10ml for blood culture
Bacterial confirmation

21. 108SHPT @Bacterial screen vs culture discrepancy
Figure 6: Difference in diagnostics of sepsis causative pathogens either by molecular approach using from direct human DNA removed samples or classical blood culture methodology: upper panel Ven diagram shows the diagnostics overlap between PCR method using human DNA removed input and blood culture, lower panel shows diagnostic details of the two methods

22. The discrepancy showed by previous studies
Blood culture(+)
Multiplex PCR (+)
67 (21,5%)
27 (8,7%)
18 (5,8%)
199 (64%)
n=311
Blood(-)
PCR (-)
Frank Bloos et al. 2012

23. In conclude:
Targeted enrichment of bacterial DNA as consequence of human DNA removal significantly enhances the sensitivity of downstream PCR based sepsis diagnostics

24. Thank you for your attention
We would like to acknowledge the funding from Vietnamese Ministry of Science and Technology (Grant: KC-10.43/11-15) for this study
Thank you for your attention