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 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