Transcript Trung Wed 1530

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
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, 2010
Classical Sepsis detection
tools
CÁC PHƯƠNG PHÁP CHẨN ĐOÁN NN GÂY NKH
Clinical
symptoms
Immune
response
monitor
Blood
culture
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
→ need to develop relevent
approaches that work
complimentary to blood culture
*NicasioMancini et al, 2010
Nucleic acid test(NAT)
PCR combined mass
spectrometric DNA
sequencing
Disadvantage:
1. Supper expensive equipments : including both PCR system
and Mass spectrometric installation
2. Well trained personnel
Promising technology, especially
for multi-readout assays
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 10- 500 CFU/ml
Klouche and Schröder 2008
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 removal
bacterial 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
Removal of 97-98% of human
DNA from blood samples
Beta-globin
6 cycles
Total DNA
prepared by
NaOH/SDS
DNA processed
by MBLC1
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
E. coli
A. baumanii
S.aureus
P. aeruginosa
K. pneumonia
S pneumonia
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
4h
Group specfic screening assays
6h
Non-Enterobacteriaceae
Gram(-)
Enterobactericeae
P. aeruginosa
A. baumannii
N. meningitidis
H. influenza
E. coli
K. pneumoniae
Salmollela
P. mirabilis
Gram(+)
Staphylococus sp
Streptococus sp
Enterococcocus sp
In real clinical diagnosis
1.2 ml for In-house
human DNA removal
Suspected
blood sepsis
10ml for blood
culture
In put DNA
Group specific
screening realtime
PCR analysis
Genus specific
realtime PCR
confirmation
114 sepsis suspected blood
samples recruited
Bacterial confirmation
108SHPT @Bacterial screen vs culture discrepancy
The discrepancy showed by previous studies
Blood
culture(+)
Multiplex PCR (+)
18 (5,8%)
27
(8,7%)
67
(21,5%)
199
(64%)
n=311
Blood(-)
PCR (-)
Frank Bloos et al. 2012
In conclude:
Targeted enrichment of bacterial DNA as consequence of
human DNA removal significantly enhances the sensitivity
of downstream PCR based sepsis diagnostics
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