dr ngo tat trung, phd danang– september 2015
DESCRIPTION
Sepsis related deaths Hospitalized sepsis patient Sepsis related death Năm US: 750.000 patients/year; 215 000 death/year, death=20-70%; rank 10th; account for 6% death. European135 000 death/ year, rank 3th of death Harrison et al. Critical Care 2006; Nicasio Mancini, 2010TRANSCRIPT
Translating molecular testing into sepsis diagnosis:
the challenge in clinical practices.
Dr Ngo Tat Trung, PhD
(Dept. Molecular biology108 Military Central Hospital)
Danang– September 2015
Harrison et al. Critical Care 2006; Nicasio Mancini, 2010
Hospitalized sepsis patient
Sepsis related death
Năm
Sepsis related deaths
US: 750.000 patients/year; 215 000 death/year, death=20-70%; rank 10th; account for 6% death.European135 000 death/ year, rank 3th of death
Clinical symptoms
Blood culture
Immune response monitor
CÁC PHƯƠNG PHÁP CHẨN ĐOÁN NN GÂY NKHClassical Sepsis detection tools
Blood culture
Classical method, widely implemented but still far from making clinician satisfied because of its intrinsic drawbacks:
1.Impractical for fastidious pathogens 2.Time required for first wave of microbial colonies to appear is too long that might switch patients into worse deleterious situation 3.Large volumes blood is mandatory for proper culturing of aerobic and anaerobic bacteria
Consequence of mis/late detection
• Risk to be sepsis shock:• Reduce chances to survive• Increased hospital cost• Drug resistance clones emerge
*NicasioMancini et al, 2010
→ need to develop relevent approaches that work complimentary to blood culture
Nucleic acid test(NAT)PCR combined mass spectrometric DNA
sequencing
Disadvantage: 1.Supper expensive equipments : including both PCR system and Mass spectrometric installation2.Well trained personnel
Promising technology, especially for multi-readout assays
Challenges to PCR’s sensitivity
Patients would present sepsis – related clinical symptoms if bacterial load exceed 10- 500 CFU/ml
Klouche and Schröder 2008
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
How the primers mis-pairing to human DNA happens
NATURE | VOL 431 | 9 SEPTEMBER 2004
Virut
Two aspects must be focused to harness PCR into sepsis diagnosis
1. Human DNA removal/bacterial DNA enrichment
2. Optimize the conditions for PCR diagnostic algorithm
Virut
Our strategic resolution
Human DNA removalbacterial DNA enrichment
Disrupt human blood cells/shear but keep bacterial cells intact
Spin to pellet bacterial cells
Total DNA extraction
Input template for PCR assays
DNA processed by MBLC1
Total DNA prepared by NaOH/SDS
MCLB1
Beta-globin
Removal of 97-98% of human DNA from blood samples
NaOH- SDS
6 cycles
Bacterial limits of detectionUpon human DNA removal
Disrupt human blood cells and shear chromatin by basic pH combined polar detergent
Pseudo sepsis samples (bacterial spiked healthy blood dilution series)
Spin to pellet bacterial cells
Total DNA extraction
Input template for PCR assays
/
E. coli
A. baumanii
S.aureus
P. aeruginosa
K. pneumonia
1 CFU/m
l
1000
0 CFU/m
l
S pneumonia
10 C
FU/ml
100 C
FU/ml
1000
CFU/m
l
Blank c
ontro
l
Salmonella
P. mirabilis
S. pidermidis
S. suise
Enterococcus sp
Enterobacteriaceae piked healthy blood dilution series
Optimize the conditions for PCR diagnostic algorithm
Input bacterial DNA extracted after human chromatin removal
Group specfic screening assays
Non-EnterobacteriaceaeGram(-)
Enterobactericeae Gram(+)
P. aeruginosaA. baumanniiN. meningitidisH. influenza
E. coli K. pneumoniaeSalmollelaP. mirabilis
Staphylococus spStreptococus sp Enterococcocus sp
4h6h
Suspected blood sepsis
1.2 ml for In-house human DNA removal In put DNA
Group specific screening realtime PCR analysis
Genus specific realtime PCR confirmation
10ml for blood culture Bacterial confirmation
In real clinical diagnosis
114 sepsis suspected blood samples recruited
108SHPT @Bacterial screen vs culture discrepancy
67 (21,5%)
27 (8,7%)
18 (5,8%)
199 (64%)
Blood culture(+)
Multiplex PCR (+)
Blood(-)
PCR (-)
Frank Bloos et al. 2012
n=311
The discrepancy showed by previous studies
In conclude: Targeted enrichment of bacterial DNA as consequence of human DNA removal significantly enhances the sensitivity of downstream PCR based sepsis diagnostics
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